optical microscopy - Olympus Microscopy Resource Center

optical microscopy - Olympus Microscopy Resource Center
OPTICAL MICROSCOPY
Davidson and Abramowitz
OPTICAL MICROSCOPY
Michael W. Davidson1 and Mortimer Abramowitz2
1
National High Magnetic Field Laboratory, The Florida State University, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32306,
[email protected], http://microscopy.fsu.edu
2
Olympus America, Inc., 2 Corporate Center Dr., Melville, New York 11747, [email protected], http://www.olympus.com
Keywords: microscopy, phase contrast, differential interference contrast, DIC, polarized light, Hoffman
modulation contrast, photomicrography, fluorescence microscopy, numerical aperture, CCD electronic cameras,
CMOS active pixel sensors, darkfield microscopy, Rheinberg illumination.
binocular microscopes with image-erecting prisms, and
the first stereomicroscope (14).
Early in the twentieth century, microscope
manufacturers began parfocalizing objectives, allowing the
image to remain in focus when the microscopist exchanged
objectives on the rotating nosepiece. In 1824, Zeiss
introduced a LeChatelier-style metallograph with infinitycorrected optics, but this method of correction would not
see widespread application for another 60 years.
Shortly before World War II, Zeiss created several
prototype phase contrast microscopes based on optical
principles advanced by Frits Zernike. Several years later
the same microscopes were modified to produce the first
time-lapse cinematography of cell division photographed
with phase contrast optics (14). This contrast-enhancing
technique did not become universally recognized until the
1950s and is still a method of choice for many cell
biologists today.
Physicist
Georges
Nomarski
introduced
improvements in Wollaston prism design for another
powerful contrast-generating microscopy theory in 1955
(15). This technique is commonly referred to as
Nomarski interference or differential interference
contrast (DIC) microscopy and, along with phase contrast,
has allowed scientists to explore many new arenas in
biology using living cells or unstained tissues. Robert
Hoffman (16) introduced another method of increasing
contrast in living material by taking advantage of phase
gradients near cell membranes. This technique is now
termed Hoffman Modulation Contrast, and is available as
optional equipment on most modern microscopes.
The majority of microscopes manufactured around the
world had fixed mechanical tube lengths (ranging from
160 to 210 millimeters) until the late 1980s, when
manufacturers largely migrated to infinity-corrected
optics. Ray paths through both finite tube length and
infinity-corrected microscopes are illustrated in Figure
1. The upper portion of the figure contains the essential
optical elements and ray traces defining the optical train
Introduction
The past decade has witnessed an enormous growth in
the application of optical microscopy for micron and submicron level investigations in a wide variety of disciplines
(reviewed in references 1-5). Rapid development of new
fluorescent labels has accelerated the expansion of
fluorescence microscopy in laboratory applications and
research (6-8). Advances in digital imaging and analysis
have also enabled microscopists to acquire quantitative
measurements quickly and efficiently on specimens
ranging from photosensitive caged compounds and
synthetic ceramic superconductors to real-time
fluorescence microscopy of living cells in their natural
environment (2, 9). Optical microscopy, with help of
digital video, can also be used to image very thin optical
sections, and confocal optical systems are now in
operation at most major research institutions (10-12).
Early microscopists were hampered by optical
aberration, blurred images, and poor lens design, which
floundered until the nineteenth century. Aberrations were
partially corrected by the mid-nineteenth century with the
introduction of Lister and Amici achromatic objectives
that reduced chromatic aberration and raised numerical
apertures to around 0.65 for dry objectives and up to 1.25
for homogeneous immersion objectives (13). In 1886,
Ernst Abbe’s work with Carl Zeiss led to the production
of apochromatic objectives based for the first time on
sound optical principles and lens design (14). These
advanced objectives provided images with reduced
spherical aberration and free of color distortions
(chromatic aberration) at high numerical apertures.
Several years later, in 1893, Professor August Köhler
reported a method of illumination, which he developed
to optimize photomicrography, allowing microscopists to
take full advantage of the resolving power of Abbe’s
objectives. The last decade of the nineteenth century saw
innovations in optical microscopy, including
metallographic microscopes, anastigmatic photolenses,
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OPTICAL MICROSCOPY
of a conventional finite tube length microscope (17). An
object (O) of height h is being imaged on the retina of
the eye at O”. The objective lens (Lob) projects a real
and inverted image of O magnified to the size O’ into the
intermediate image plane of the microscope. This occurs
at the eyepiece diaphragm, at the fixed distance fb + z’
behind the objective. In this diagram, fb represents the
back focal length of the objective and z’ is the optical tube
length of the microscope. The aerial intermediate image
at O’ is further magnified by the microscope eyepiece
(Ley) and produces an erect image of the object at O” on
the retina, which appears inverted to the microscopist.
The magnification factor of the object is calculated by
considering the distance (a) between the object (O) and
the objective (Lob) , and the front focal length of the
objective lens (f). The object is placed a short distance
(z) outside of the objective’s front focal length (f), such
that z + f = a. The intermediate image of the object, O’, is
located at distance b, which equals the back focal length
of the objective (fb) plus (z’), the optical tube length of
the microscope. Magnification of the object at the
intermediate image plane equals h’. The image height at
this position is derived by multiplying the microscope
tube length (b) by the object height (h), and dividing this
by the distance of the object from the objective: h’ = (h x
b)/a. From this argument, we can conclude that the lateral
or transverse magnification of the objective is equal to a
factor of b/a (also equal to f/z and z’/fb), the back focal
length of the objective divided by the distance of the object
from the objective. The image at the intermediate plane
(h’) is further magnified by a factor of 25 centimeters
(called the near distance to the eye) divided by the focal
length of the eyepiece. Thus, the total magnification of
the microscope is equal to the magnification by the
objective times that of the eyepiece. The visual image
(virtual) appears to the observer as if it were 10 inches
away from the eye.
Most objectives are corrected to work within a narrow
range of image distances, and many are designed to work
only in specifically corrected optical systems with
matching eyepieces. The magnification inscribed on the
objective barrel is defined for the tube length of the
microscope for which the objective was designed.
The lower portion of Figure 1 illustrates the optical
train using ray traces of an infinity-corrected microscope
system. The components of this system are labeled in a
similar manner to the finite-tube length system for easy
comparison. Here, the magnification of the objective is
the ratio h’/h, which is determined by the tube lens (Ltb).
Note the infinity space that is defined by parallel light
beams in every azimuth between the objective and the tube
lens. This is the space used by microscope manufacturers
to add accessories such as vertical illuminators, DIC
Davidson and Abramowitz
Figure 1. Optical trains of finite-tube and infinity-corrected
microscope systems. (Upper) Ray traces of the optical train
representing a theoretical finite-tube length microscope. The object
(O) is a distance (a) from the objective (Lob) and projects an
intermediate image (O’) at the finite tube length (b), which is further
magnified by the eyepiece (Ley) and then projected onto the retina
at O’’. (Lower) Ray traces of the optical train representing a
theoretical infinity-corrected microscope system.
prisms, polarizers, retardation plates, etc., with much
simpler designs and with little distortion of the image
(18). The magnification of the objective in the infinitycorrected system equals the focal length of the tube lens
divided by the focal length of the objective.
Fundamentals of Image Formation
In the optical microscope, when light from the
microscope lamp passes through the condenser and then
through the specimen (assuming the specimen is a light
absorbing specimen), some of the light passes both around
and through the specimen undisturbed in its path. Such
light is called direct light or undeviated light. The
background light (often called the surround) passing
around the specimen is also undeviated light.
Some of the light passing through the specimen is
deviated when it encounters parts of the specimen. Such
deviated light (as you will subsequently learn, called
diffracted light) is rendered one-half wavelength or 180
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OPTICAL MICROSCOPY
degrees out of step (more commonly, out of phase) with
the direct light that has passed through undeviated. The
one-half wavelength out of phase, caused by the specimen
itself, enables this light to cause destructive interference
with the direct light when both arrive at the intermediate
image plane located at the fixed diaphragm of the
eyepiece. The eye lens of the eyepiece further magnifies
this image which finally is projected onto the retina, the
film plane of a camera, or the surface of a light-sensitive
computer chip.
What has happened is that the direct or undeviated light
is projected by the objective and spread evenly across the
entire image plane at the diaphragm of the eyepiece. The
light diffracted by the specimen is brought to focus at
various localized places on the same image plane, where
the diffracted light causes destructive interference and
reduces intensity resulting in more or less dark areas.
These patterns of light and dark are what we recognize as
an image of the specimen. Because our eyes are sensitive
to variations in brightness, the image becomes a more or
less faithful reconstitution of the original specimen.
To help understand the basic principles, it is suggested
that readers try the following exercise and use as a
specimen an object of known structure, such as a stage
micrometer or similar grating of closely spaced dark
lines. To proceed, place the finely ruled grating on the
microscope stage and bring it into focus using first a 10x
and then the 40x objective (18). Remove the eyepiece
and, in its place, insert a phase telescope so the rear focal
plane of the objective can be observed. If the condenser
aperture diaphragm is closed most of the way, a bright
white central spot of light will appear at the back of the
objective, which is the image of the aperture diaphragm.
To the right and left of the central spot, a series of spectra
(also images of the aperture diaphragm) will be present,
each colored blue on the part closest to the central spot
and colored red on the part of the spectrum farthest from
the central bright spot (as illustrated in Figure 2). The
intensity of these colored spectra decreases according
to how far the spectrum is from the central spot (17,18).
Those spectra nearer the periphery of the objective
are dimmer than those closer to the central spot. The
diffraction spectra illustrated in Figure 2 using three
different magnifications. In Figure 2(b), the diffraction
pattern visible at the rear focal plane of the 10X objective
contains two diffraction spectra. If the grating is removed
from the stage, as illustrated in Figure 2(a), these spectra
disappear and only the central image of the aperture
diaphragm remains. If the grating is reinserted, the spectra
reappear once again. Note that the spaces between the
colored spectra appear dark. Only a single pair of spectra
can be observed if the grating is examined with the 10x
objective. In this case, one diffraction spot appears to
Davidson and Abramowitz
the left and one appears to the right of the central aperture
opening. If the line grating is examined with a 40x
objective (as shown in Figure 2(c)), several diffraction
spectra appear to the left and right of the central aperture.
When the magnification is increased to 60x (and assuming
it has a higher numerical aperture than the 40x objective),
additional spectra (Figure 2(d)) appear to the right and
left than are visible with the 40x objective in place.
Because the colored spectra disappear when the
grating is removed, it can be assumed that it is the
specimen itself that is affecting the light passing through,
thus producing the colored spectra. Further, if the aperture
diaphragm is closed down, we will observe that objectives
of higher numerical aperture grasp more of these colored
spectra than do objectives of lower numerical aperture.
The crucial importance of these two statements for
understanding image formation will become clear in the
ensuing paragraphs.
Figure 2. Diffraction spectra seen at the rear focal plane of the
objective through a focusing telescope when imaging a closely
spaced line grating. (a) Image of the condenser aperture diaphragm
with an empty stage. (b) Two diffraction spectra from a 10x
objective when a finely ruled line grating is placed on the
microscope stage. (c) Diffraction spectra of the line grating from
a 40x objective. (d) Diffraction spectra of the line grating from a
60x objective.
The central spot of light (image of the condenser
aperture diaphragm) represents the direct or undeviated
light passing through the specimen or around the specimen
undisturbed (illustrated in Figure 3(b)). It is called the
0th or zeroth order. The fainter images of the aperture
diaphragm on each side of the zeroth order are called the
1st, 2nd, 3rd, 4th, etc. orders respectively, as represented
by the simulated diffraction pattern in Figure 3(a) that
would be observed at the rear focal plane of a 40x
objective. All the captured orders represent, in this case,
the diffraction pattern of the line grating as seen at the
rear focal plane of the objective (18).
The fainter diffracted images of the aperture
diaphragm are caused by light deviated or diffracted, spread
out in fan shape, at each of the openings of the line grating
(Figure 3(b)). The blue wavelengths are diffracted at a
lesser angle than the green wavelengths, which are
diffracted at a lesser angle than the red wavelengths.
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OPTICAL MICROSCOPY
Davidson and Abramowitz
At the rear focal plane of the objective, the blue
wavelengths from each slit interfere constructively to
produce the blue area of the diffracted image of each
spectrum or order; similarly for the red and green areas
(Figure 3(a)). Where the diffracted wavelengths are 1/2
wave out of step for each of these colors, the waves
destructively interfere. Hence the dark areas between the
spectra or orders. At the position of the zeroth order, all
wavelengths from each slit add constructively. This
produces the bright white light you see as the zeroth order
at the center of the rear focal plane of the objective
(Figures 2, 3 and 4).
Figure 4. Diffraction patterns generated by narrow and wide
slits and by complex grids. (a) Conoscopic image of the grid seen
at the rear focal plane of the objective when focused on the wide
slit pattern in (b). (b) Orthoscopic image of the grid with greater
slit width at the top and lesser width at the bottom. (c) Conoscopic
image of the narrow width portion of the grid (lower portion of
(b)). (d) and (f) Orthoscopic images of grid lines arranged in a
square pattern (d) and a hexagonal pattern (f). (e) and (g)
Conoscopic images of patterns in (d) and (f), respectively.
at the rear focal plane of the objective (Figure 4(e).
Likewise, the orthoscopic image of a hexagonally arranged
grid (Figure 4(f)) produces a corresponding hexagonally
arranged conoscopic image of first order diffraction
patterns (Figure 4(g)).
Microscope specimens can be considered as complex
gratings with details and openings of various sizes. This
concept of image formation was largely developed by
Ernst Abbe, the famous German microscopist and optics
theoretician of the 19th century. According to Abbe (his
theories are widely accepted at the present time), the
details of a specimen will be resolved if the objective
captures the 0th order of the light and at least the 1st order
(or any two orders, for that matter). The greater the
number of diffracted orders that gain admittance to the
objective, the more accurately the image will represent
the original object (2, 14, 17, 18).
Further, if a medium of higher refractive index than
air (such as immersion oil) is used in the space between
the front lens of the objective and the top of the cover
slip (as shown in Figure 5(a)), the angle of the diffracted
orders is reduced and the fans of diffracted light will be
compressed. As a result, an oil immersion objective can
capture more diffracted orders and yield better resolution
than a dry objective (Figure 5(b)). Moreover, because
blue light is diffracted at a lesser angle than green light or
red light, a lens of a given aperture may capture more
orders of light when the wavelengths are in the blue region
of the visible light spectrum. These two principles explain
Figure 3. Diffraction spectra generated at the rear focal plane
of the objective by undeviated and diffracted light. (a) Spectra
visible through a focusing telescope at the rear focal plane of a
40x objective. (b) Schematic diagram of light both diffracted and
undeviated by a line grating on the microscope stage.
The closer the spacing of a line grating, the fewer the
spectra that will be captured by a given objective, as
illustrated in Figure 4(a-c). The diffraction pattern
illustrated in Figure 4(a) was captured by a 40x objective
imaging the lower portion the line grating in Figure 4(b),
where the slits are closer together (17, 18). In Figure
4(c), the objective is focused on the upper portion of the
line grating (Figure 4(b)) where the slits are farther apart,
and more spectra are captured by the objective. The direct
light and the light from the diffracted orders continue on,
being focused by the objective, to the intermediate image
plane at the fixed diaphragm of the eyepiece. Here the
direct and diffracted light rays interfere and are thus
reconstituted into the real, inverted image that is seen by
the eye lens of the eyepiece and further magnified. This
is illustrated in Figure 4 (d-g) with two types of
diffraction gratings. The square grid illustrated in Figure
4(d) represents the orthoscopic image of the grid (i.e.
the usual specimen image) as seen through the full aperture
of the objective. The diffraction pattern derived from this
grid is shown as a conoscopic image that would be seen
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OPTICAL MICROSCOPY
the classic Rayleigh equation often cited for resolution
(2, 18-20):
d = 1.22 (l / 2NA)
(1)
Davidson and Abramowitz
of the objective can have significant effect upon the
eventual image produced (18). For small details in a
specimen (rather than a grating), the objective projects
the direct and diffracted light onto the image plane of the
eyepiece diaphragm in the form of small, circular
diffraction disks known as Airy disks (illustrated in Figure
6). High numerical aperture objectives capture more of
the diffracted orders and produce smaller size disks than
do low numerical aperture objectives. In Figure 6, Airy
disk size is shown steadily decreasing from Figure 6(a)
Where d is the space between two adjacent particles (still
allowing the particles to be perceived as separate), l is
the wavelength of illumination, and NA is the numerical
aperture of the objective.
Figure 5. Effect of imaging medium refractive index on diffracted
orders captured by the objective. (a) Conoscopic image of objective
back focal plane diffraction spectra when air is the medium between
the cover slip and the objective front lens. (b) Diffraction spectra
when immersion oil of refractive index similar to glass is used in
the space between the cover slip and the objective front lens.
The greater the number of higher diffracted orders
admitted into the objective, the smaller the details of the
specimen that can be clearly separated (resolved). Hence
the value of using high numerical aperture for such
specimens. Likewise, the shorter the wavelength of visible
light used, the better the resolution. These ideas explain
why high numerical aperture, apochromatic lenses can
separate extremely small details in blue light.
Placing an opaque mask at the back of the objective
blocks the outermost diffracted orders. This either
reduces the resolution of the grating lines, or any other
object details, or it destroys the resolution altogether so
that the specimen is not visible. Hence the usual caution
not to close down the condenser aperture diaphragm
below the suggested 2/3 to 9/10 of the objective’s
aperture.
Failure of the objective to grasp any of the diffracted
orders results in an unresolved image. In a specimen with
very minute details, the diffraction fans are spread at a
very large angle, requiring a high numerical aperture
objective to capture them. Likewise, because the
diffraction fans are compressed in immersion oil or in
water, objectives designed for such use can give better
resolution than dry objectives.
If alternate diffracted orders are blocked out (still
assuming the grating as our specimen), the number of lines
in the grating will appear doubled (a spurious resolution).
The important caveat is that actions introduced at the rear
Figure 6. Airy disks and resolution. (a-c) Airy disk size and
related intensity profile (point spread function) as related to
objective numerical aperture, which decreases from (a) to (c) as
numerical aperture increases. (e) Two Airy disks so close together
that their central spots overlap. (d) Airy disks at the limit of
resolution.
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OPTICAL MICROSCOPY
Davidson and Abramowitz
imperfections on the glass surfaces of the condenser. The
opening size of the condenser aperture diaphragm, along
with the aperture of the objective, determines the realized
numerical aperture of the microscope system. As the
condenser diaphragm is opened, the working numerical
aperture of the microscope increases, resulting in greater
light transmittance and resolving power. Parallel light rays
that pass through and illuminate the specimen are brought
to focus at the rear focal plane of the objective, where
the image of the variable condenser aperture diaphragm
and the light source are observed in focus simultaneously.
through Figure 6(c). The larger disk sizes in Figures 6(a)
and (b) are produced by objectives with lower numerical
aperture, while the very sharp Airy disk in Figure 6(c) is
produced by an objective of very high numerical aperture
(2, 18).
The resulting image at the eyepiece diaphragm level
is actually a mosaic of Airy disks which are perceived as
light and dark regions of the specimen. Where two disks
are so close together that their central black spots overlap
considerably, the two details represented by these
overlapping disks are not resolved or separated and thus
appear as one (illustrated in Figure 6(d)). The Airy disks
shown in Figure 6(e) are just far enough apart to be
resolved.
The basic principle to be remembered is that the
combination of direct and diffracted light (or the
manipulation of direct or diffracted light) is critically
important in image formation. The key places for such
manipulation are the rear focal plane of the objective and
the front focal plane of the substage condenser. This
principle is fundamental to most of the contrast
improvement methods in optical microscopy (18, and see
the section on Contrast Enhancing Techniques); it is
of particular importance at high magnification of small
details close in size to the wavelength of light. Abbe was
a pioneer in developing these concepts to explain image
formation of light-absorbing or amplitude specimens (2,
18-20).
Köhler Illumination
Proper illumination of the specimen is crucial in
achieving high-quality images in microscopy and critical
photomicrography. An advanced procedure for
microscope illumination was first introduced in 1893 by
August Köhler, of the Carl Zeiss corporation, as a method
of providing optimum specimen illumination. All
manufacturers of modern laboratory microscopes
recommend this technique because it produces specimen
illumination that is uniformly bright and free from glare,
thus allowing the user to realize the microscope’s full
potential.
Most modern microscopes are designed so that the
collector lens and other optical components built into the
base will project an enlarged and focused image of the
lamp filament onto the plane of the aperture diaphragm
of a properly positioned substage condenser. Closing or
opening the condenser diaphragm controls the angle of
the light rays emerging from the condenser and reaching
the specimen from all azimuths. Because the light source
is not focused at the level of the specimen, illumination
at specimen level is essentially grainless and extended,
and does not suffer deterioration from dust and
Figure 7. Light paths in Kohler illumination. The illuminating ray
paths are illustrated on the left side and the image-forming ray
paths on the right. Light emitted from the lamp passes through a
collector lens and then through the field diaphragm. The aperture
diaphragm in the condenser determines the size and shape of the
illumination cone on the specimen plane. After passing through
the specimen, light is focused at the back focal plane of the objective
and then proceeds to and is magnified by the ocular before passing
into the eye.
Light pathways illustrated in Figure 7 are schematically
drawn to represent separate paths taken by the specimenilluminating light rays and the image forming light rays
(17). This is not a true representation of any real
segregation of these pathways, but a diagrammatic
representation presented for purposes of visualization and
discussion. The left-hand diagram in Figure 7
demonstrates that the ray paths of illuminating light
produce a focused image of the lamp filament at the plane
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OPTICAL MICROSCOPY
of the substage condenser aperture diaphragm, the rear
focal plane of the objective, and the eyepoint (also called
the Ramsden disk) above the eyepiece. These areas in
common focus are often referred to as conjugate planes,
a principle that is critical in understanding the concept of
Köhler illumination (2, 17-21). By definition, an object
that is in focus at one plane is also in focus at other
conjugate planes of that light path. In each light pathway
(both image forming and illumination), there are four
separate planes that together make up a conjugate plane
set.
Conjugate planes in the path of the illuminating light
rays in Köhler illumination (left-hand diagram in Figure
7) include the lamp filament, condenser aperture
diaphragm (at the front focal plane of the condenser), the
rear focal plane of the objective, and the eyepoint of the
eyepiece. The eyepoint is located approximately one-half
inch (one centimeter) above the top lens of the eyepiece,
at the point where the observer places the front of the eye
during observation.
Likewise, the conjugate planes in the image-forming
light path in Köhler illumination (right-hand diagram in
Figure 7) include the field diaphragm, the focused
specimen, the intermediate image plane (i.e., the plane of
the fixed diaphragm of the eyepiece), and the retina of
the eye or the film plane of the camera. The presence of
conjugate focal planes is often useful in troubleshooting
a microscope for contaminating dust, fibers, and
imperfections in the optical elements. When such artifacts
are in sharp focus, it follows that they must reside on or
near a surface that is part of the imaging-forming set of
conjugate planes. Members of this set include the glass
element at the microscope light port, the specimen, and
the graticule (if any) in the eyepiece. Alternatively, if
these contaminants are out of focus, then they occur near
the illuminating set of elements that share conjugate
planes. Suspects in this category are the condenser top
lens (where dust and dirt often accumulate), the exposed
eyepiece lens element (contaminants from eyelashes), and
the objective front lens (usually fingerprint smudges).
In Köhler illumination, light emitted from the
tungsten-halide lamp filament first passes through a
collector lens located close to the lamp housing, and then
through a field lens that is near the field diaphragm. A
sintered or frosted glass filter is often placed between
the lamp and the collector lens to diffuse the light and
ensure an even intensity of illumination. In this case, the
image of the lamp filament is focused onto the front focal
plane of the condenser while the diffuser glass is
temporarily removed from the light path. The focal length
of the collector lens must be carefully matched to the
lamp filament dimensions to ensure that a filament image
of the appropriate size is projected into the condenser
Davidson and Abramowitz
aperture. For proper Köhler illumination, the image of
the filament should completely fill the condenser
aperture.
The field lens is responsible for bringing the image
of the filament into focus at the plane of the substage
condenser aperture diaphragm. A first surface mirror
(positioned at a 45-degree angle to the light path) reflects
focused light leaving the field lens through the field
diaphragm and into the substage condenser. The field
diaphragm iris opening serves as a virtual light source for
the microscope, and its image is focused by the condenser
(raised or lowered) onto the specimen plane. Optical
designs for the arrangement of these elements may vary
by microscope manufacturer, but the field diaphragm
should be positioned at a sufficient distance from the field
lens to eliminate dust and lens imperfections from being
imaged in the plane of the specimen.
The field diaphragm in the base of the microscope
controls only the width of the bundle of light rays reaching
the condenser—it does not affect the optical resolution,
numerical aperture, or the intensity of illumination.
Proper adjustment of the field diaphragm (i.e., focused
by adjusting the height of the condenser and centered in
the optical path, then opened so as to lie just outside of
the field of view) is important for preventing glare, which
can reduce contrast in the observed image. The
elimination of unwanted light is particularly important
when attempting to image specimens with inherently low
contrast. When the field diaphragm is opened too far,
scattered light originating from the specimen and light
reflected at oblique angles from optical surfaces can act
to degrade image quality.
The substage condenser is typically mounted directly
beneath the microscope stage in a bracket that can be
raised or lowered independently of the stage. Control of
the aperture diaphragm opening size occurs with either a
swinging arm, a lever, or by rotating a collar on the
condenser housing. The most critical aspect of achieving
proper Köhler illumination is correct adjustment of the
substage condenser. Condenser misalignment and an
improperly adjusted condenser aperture diaphragm are the
main sources of image degradation and poor quality
photomicrography (19).
When properly adjusted, light from the condenser will
fill the rear focal plane of the objective and project a cone
of light into the field of view. The condenser aperture
diaphragm is responsible for controlling the angle of the
illuminating light cone and, consequently, the working
numerical aperture of the condenser. It is important to
note, with respect to the size and shape of condenser light
cones, that reducing the size of the field diaphragm only
serves to slightly decrease the size of the lower portions
of the light cone. The angle and numerical aperture of
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OPTICAL MICROSCOPY
the light cone remains essentially unchanged with
reduction in field diaphragm size (21). Illumination
intensity should not be controlled through opening and
closing the condenser aperture diaphragm, or by shifting
the condenser up and down or axially with respect to the
optical center of the microscope. It should only be
controlled through the use of neutral density filters placed
into the light path or by reducing voltage to the lamp
(although the latter is not usually recommended, especially
for photomicrography). To ensure the maximum
performance of the tungsten-halide lamp, refer to the
manufacturer’s instrument manual to determine the
optimum lamp voltage (usually 5-10 volts) and use that
setting. Adding or removing neutral density filters can
then easily control brightness of the illumination without
affecting color temperature.
The size of the substage condenser aperture diaphragm
opening should not only coincide with the desired
numerical aperture, but also the quality of the resulting
image should be considered. In general, the diaphragm
should be set to a position that allows 2/3 to 9/10 (60 to
90 percent) of the entire light disc size (visible at the
rear focal plane of the objective after removal of the
eyepiece or with a Bertrand lens). These values may vary
with extremes in specimen contrast.
The condenser aperture diaphragm should be set to an
opening size that will provide a compromise of resolution
and contrast that depends, to a large degree, on the
absorption, diffraction, and refraction characteristics of
the specimen. This adjustment must be accomplished
without overwhelming the image with artifacts that
obscure detail and present erroneous enhancement of
contrast. The amount of image detail and contrast
necessary to produce the best photomicrograph is also
dependent upon refractive index, optical characteristics,
and other specimen-dependent parameters.
When the aperture diaphragm is erroneously closed
too far, resulting diffraction artifacts cause visible fringes,
banding, and/or pattern formation in photomicrographs.
Other problems, such as refraction phenomena, can also
produce apparent structures in the image that are not real
(21). Alternatively, opening the condenser aperture too
wide causes unwanted glare and light scattering from the
specimen and optical surfaces within the microscope,
leading to a significant loss of contrast and washing out
of image detail. The correct setting will vary from
specimen to specimen, and the experienced microscopist
will soon learn to accurately adjust the condenser aperture
diaphragm (and numerical aperture of the system) by
observing the image without necessarily having to view
the diaphragm in the rear focal plane of the objective. In
fact, many microscopists (including the authors) believe
that critical adjustment of the numerical aperture of the
Davidson and Abramowitz
microscope system to optimize image quality is the single
most important step in photomicrography.
The illumination system of the microscope, when
adjusted for proper Köhler illumination, must satisfy
several requirements. The illuminated area of the
specimen plane must no larger than the field of view for
any given objective/eyepiece combination. Also, the light
must be of uniform intensity and the numerical aperture
may vary from a maximum (equal to that of the objective)
to a lesser value that will depend upon the optical
characteristics of the specimen. Table 1 contains a list
of objective numerical apertures versus the field of view
diameter (for an eyepiece of field number 22 with no tube
lens present – see discussion on field number) for each
objective, ranging from very low to very high
magnifications.
Many microscopes are equipped with specialized
substage condensers that have a swing-out top lens, which
can be removed from the optical path for use with lower
Table 1 Viewfield Diameters (FN 22)
(SWF 10x Eyepiece)
Objective
Magnification
1/2x
1x
2x
4x
10x
20x
40x
50x
60x
100x
150x
250x
a
Diameter
(mm)
44.0
22.0
11.0
5.5
2.2
1.1
0.55
0.44
0.37
0.22
0.15
0.088
Source: Nikon
power objectives (2x through 5x). This action changes
the performance of the remaining components in the light
path, and some adjustment is necessary to achieve the best
illumination conditions. The field diaphragm can no
longer be used for alignment and centering of the substage
condenser and is now ineffective in limiting the area of
the specimen under illumination. Also, much of the
unwanted glare once removed by the field diaphragm is
reduced because the top lens of the condenser produces
a light cone having a much lower numerical aperture,
allowing light rays to pass through the specimen at much
lower angles. Most important, the optical conditions for
8
OPTICAL MICROSCOPY
Köhler illumination no longer apply.
For low power objectives (2x to 5x), alignment of the
microscope optical components and the establishment of
Köhler illumination conditions should always be
undertaken at a higher (10x) magnification before
removing the swing-out condenser lens for work at lower
(5x and below) magnifications. The height of the
condenser should then not be changed. Condenser
performance is radically changed when the swing-out lens
is removed (18, 21). The image of the lamp filament is
no longer formed in the aperture diaphragm, which ceases
to control the numerical aperture of the condenser and
the illumination system. In fact, the aperture diaphragm
should be opened completely to avoid vignetting, a gradual
fading of light at the edges of the viewfield.
Contrast adjustment in low magnification microscopy
is then achieved by adjustment of the field diaphragm (18,
19, 21). When the field diaphragm is wide open (greater
than 80 percent), specimen details are washed out and a
significant amount of scattering and glare is present.
Closing the field diaphragm to a position between 50 and
80 percent will yield the best compromise on specimen
contrast and depth of field. This adjustment is now visible
at the rear focal plane of the objective when the eyepiece
is removed or a Bertrand lens is inserted into the eye tube.
Objectives designed for low magnification are
significantly simpler in design than their higher
magnification counterparts. This is due to the smaller
angles of illuminating light cones produced by low
magnification condensers, which require objectives of
lower numerical aperture.
Measurement graticules, which must be in sharp focus
and simultaneously superimposed on the specimen image,
can be inserted into any of several conjugate planes in the
image-forming path. The most common eyepiece (ocular)
measuring and photomicrography graticules are placed in
the intermediate image plane, which is positioned at the
fixed diaphragm within the eyepiece. It is theoretically
possible to also place graticules in any image-forming
conjugate plane or in the plane of the illuminated field
diaphragm. Stage micrometers are specialized graticules
placed on microslides, which are used to calibrate
eyepiece graticules and to make specimen measurements.
Color and neutral density filters are often placed in
the optical pathway to reduce light intensity or alter the
color characteristics of the illumination. There are several
locations within the microscope stand where these filters
are usually placed. Some modern laboratory microscopes
have a filter holder sandwiched between the lamp housing
and collector lens, which serves as an ideal location for
these filters. Often, neutral density filters along with
color correction filters and a frosted diffusion filter are
placed together in this filter holder. Other microscope
Davidson and Abramowitz
designs provide a set of filters built internally into the
body, which can be toggled into the light path by means of
levers. A third common location for filters is a holder
mounted on the bottom of the substage condenser, below
the aperture diaphragm, that will accept gelatin or glass
filters.
It is important not to place filters in or near any of the
image-forming conjugate planes to avoid dirt or surface
imperfections on the filters being imaged along with the
specimen (22). Some microscopes have an attachment
for placing filters near the light port at the base (near the
field diaphragm). This placement is probably too close
to the field diaphragm, and surface contamination may be
either in sharp focus or appear as blurred artifacts
superimposed onto the image. It is also not wise to place
filters directly on the microscope stage for the same
reasons.
Microscope Objectives, Eyepieces,
Condensers, and Optical Aberrations
Finite microscope objectives are designed to project
a diffraction-limited image at a fixed plane (the
intermediate image plane) that is dictated by the
microscope tube length and located at a pre-specified
distance from the rear focal plane of the objective.
Specimens are imaged at a very short distance beyond the
front focal plane of the objective through a medium of
defined refractive index, usually air, water, glycerin, or
specialized immersion oils. Microscope manufacturers
offer a wide range of objective designs to meet the
performance needs of specialized imaging methods (2,
6, 9, 18-21, and see the section on Contrast Enhancing
Techniques), to compensate for cover glass thickness
variations, and to increase the effective working distance
of the objective.
All of the major microscope manufacturers have now
changed their design to infinity-corrected objectives.
Such objectives project emerging rays in parallel bundles
from every azimuth to infinity. They require a tube lens
in the light path to bring the image into focus at the
intermediate image plane.
The least expensive (and most common) objectives
are the achromatic objectives, which are corrected for
axial chromatic aberration in two wavelengths (red and
blue) that are brought into the same focus. Further, they
are corrected for spherical aberration in the color green,
as described in Table 2. The limited correction of
achromatic objectives leads to problems with color
microscopy and photomicrography. When focus is chosen
in the red-blue region of the spectrum, images will have a
green halo (often termed residual color). Achromatic
9
OPTICAL MICROSCOPY
Table 2
Objective Lens Types and Corrections
almost eliminating chromatic aberration, and are corrected
spherically for two colors. Apochromatic objectives are
the best choice for color photomicrography in white light.
Because of their high level of correction, apochromat
objectives usually have, for a given magnification, higher
numerical apertures than do achromats or fluorites. Many
of the newer high-end fluorite and apochromat objectives
are corrected for four colors chromatically and four
colors spherically.
All three types of objectives suffer from pronounced
field curvature and project images that are curved rather
than flat. To overcome this inherent condition, lens
designers have produced flat-field corrected objectives
that yield flat images. Such lenses are called plan
achromats, plan fluorites, or plan apochromats, and
although this degree of correction is expensive, these
objectives are now in routine use due to their value in
photomicrography.
a
Corrections for Aberrations
Type
Achromat
Plan Achromat
Fluorite
Plan Fluorite
Plan Apochromat
Spherical
Chromatic
Flatness
Correction
*b
*b
3d
3d
4e
2c
2c
<3d
<3d
>4e
No
Yes
No
Yes
Yes
Davidson and Abramowitz
a
Source: Nikon Instrument Group
Corrected for two wavelengths at two specific aperture angles.
c
Corrected for blue and red - broad range of the visible spectrum.
d
Corrected for blue, green and red - full range of the visible spectrum.
e
Corrected for dark blue, blue, green and red.
b
objectives yield their best results with light passed through
a green filter (often an interference filter) and using black
and white film when these objectives are employed for
photomicrography. The lack of correction for flatness
of field (or field curvature) further hampers achromat
objectives. In the past few years, most manufacturers have
begun providing flat field corrections for achromat
objectives and have given these corrected objectives the
name of plan achromats.
The next higher level of correction and cost is found
in objectives called fluorites or semi-apochromats
illustrated by the center objective in Figure 8. This figure
depicts three major classes of objectives: The achromats
with the least amount of correction, as discussed above;
the fluorites (or semi-apochromats) that have additional
spherical corrections; and, the apochromats that are the
most highly corrected objectives available. Fluorite
objectives are produced from advanced glass formulations
that contain materials such as fluorspar or newer synthetic
substitutes (5). These new formulations allow for greatly
improved correction of optical aberration. Similar to the
achromats, the fluorite objectives are also corrected
chromatically for red and blue light. In addition, the
fluorites are also corrected spherically for two colors.
The superior correction of fluorite objectives compared
to achromats enables these objectives to be made with a
higher numerical aperture, resulting in brighter images.
Fluorite objectives also have better resolving power than
achromats and provide a higher degree of contrast, making
them better suited than achromats for color
photomicrography in white light.
The highest level of correction (and expense) is found
in apochromatic objectives, which are corrected
chromatically for three colors (red, green, and blue),
Figure 8. Levels of optical correction for aberration in commercial
objectives. (a) Achromatic objectives, the lowest level of correction,
contain two doublets and a single front lens; (b) Fluorites or semiapochromatic objectives, a medium level of correction, contain
three doublets, a meniscus lens, and a single front lens; and (c)
Apochromatic objectives, the highest level of correction, contain a
triplet, two doublets, a meniscus lens, and a single hemispherical
front lens.
Uncorrected field curvature is the most severe
aberration in higher power fluorite and apochromat
objectives, and it was tolerated as an unavoidable artifact
for many years. During routine use, the viewfield would
have to be continuously refocused between the center and
the edges to capture all specimen details. The introduction
of flat-field (plan) correction to objectives perfected their
use for photomicrography and video microscopy, and
today these corrections are standard in both general use
and high-performance objectives. Correction for field
10
OPTICAL MICROSCOPY
curvature adds a considerable number of lens elements
to the objective, in many cases as many as four additional
lenses. This significant increase in the number of lens
elements for plan correction also occurs in already
overcrowded fluorite and apochromat objectives,
frequently resulting in a tight fit of lens elements within
the objective barrel (4, 5, 18).
Before the transition to infinity-corrected optics, most
objectives were specifically designed to be used with a
set of oculars termed compensating eyepieces. An
example is the former use of compensating eyepieces with
highly corrected high numerical aperture objectives to
help eliminate lateral chromatic aberration.
There is a wealth of information inscribed on the
barrel of each objective, which can be broken down into
several categories (illustrated in Figure 9). These include
the linear magnification, numerical aperture value, optical
corrections, microscope body tube length, the type of
medium the objective is designed for, and other critical
factors in deciding if the objective will perform as needed.
Additional information is outlined below (17):
Davidson and Abramowitz
Ultrafluar (fluorite objective with glass that is
transparent down to 250 nanometers), and CF and CFI
(chrome-free; chrome-free infinity).
· Numerical Aperture: This is a critical value that
indicates the light acceptance angle, which in turn
determines the light gathering power, the resolving power,
and depth of field of the objective. Some objectives
specifically designed for transmitted light fluorescence
and darkfield imaging are equipped with an internal iris
diaphragm that allows for adjustment of the effective
numerical aperture. Designation abbreviations for these
objectives include I, Iris, W/Iris.
· Mechanical Tube Length: This is the length of the
microscope body tube between the nosepiece opening,
where the objective is mounted, and the top edge of the
observation tubes where the oculars (eyepieces) are
inserted. Tube length is usually inscribed on the objective
as the size in number of millimeters (160, 170, 210, etc.)
for fixed lengths, or the infinity symbol ( ∞) for infinitycorrected tube lengths.
Figure 10. Objective with three lens groups and correction collar
for varying cover glass thicknesses. (a) Lens group 2 rotated to
the forward position within the objective. This position is used for
the thinnest cover slips. (b) Lens group 2 rotated to the rearward
position within the objective. This position is used for the thickest
coverslips.
Figure 9. Specifications engraved on the barrel of a typical
microscope objective. These include the manufacturer, correction
levels, magnification, numerical aperture, immersion requirements,
tube length, working distance, and specialized optical properties.
· Cover Glass Thickness: Most transmitted light
objectives are designed to image specimens that are
covered by a cover glass (or cover slip). The thickness
of these small glass plates is now standardized at 0.17
mm for most applications, although there is some variation
in thickness within a batch of cover slips. For this reason,
some of the high numerical aperture dry objectives have
a correction collar adjustment of the internal lens
elements to compensate for this variation (Figure 10).
Abbreviations for the correction collar adjustment include
Corr, w/Corr, and CR, although the presence of a
movable, knurled collar and graduated scale is also an
· Optical Corrections: These are usually abbreviated
as Achro (achromat), Apo (apochromat), and Fl, Fluar,
Fluor, Neofluar, or Fluotar (fluorite) for better
spherical and chromatic corrections, and as Plan, Pl, EF,
Acroplan, Plan Apo or Plano for field curvature
corrections. Other common abbreviations are: ICS
(infinity corrected system) and UIS (universal infinity
system), N and NPL (normal field of view plan),
11
Davidson and Abramowitz
OPTICAL MICROSCOPY
indicator of this feature.
· Working Distance: This is the distance between the
objective front lens and the top of the cover glass when
the specimen is in focus. In most instances, the working
distance of an objective decreases as magnification
increases. Working distance values are not included on
all objectives and their presence varies depending upon
the manufacturer. Common abbreviations are: L, LL, LD,
and LWD (long working distance); ELWD (extra-long
working distance); SLWD (super-long working distance),
and ULWD (ultra-long working distance).
· Objective Screw Threads: The mounting threads
on almost all objectives are sized to standards of the Royal
Microscopical Society (RMS) for universal compatibility.
This standard specifies mounting threads that are 20.32
mm in diameter with a pitch of 0.706, which is currently
used in the production of infinity-corrected objectives
by manufacturers Olympus and Zeiss. Leica and Nikon
have broken from the standard with the introduction of
new infinity-corrected objectives that have a wider
mounting thread size, making Leica and Nikon objectives
usable only on their own microscopes. Abbreviations
commonly used are: RMS (Royal Microscopical Society
objective thread), M25 (metric 25-mm objective thread),
and M32 (metric 32-mm objective thread).
· Immersion Medium: Most objectives are designed
to image specimens with air as the medium between the
objective and the cover glass. To attain higher working
numerical apertures, many objectives are designed to
image the specimen through another medium that reduces
refractive index differences between glass and the imaging
medium. High-resolution plan apochromat objectives can
achieve numerical apertures up to 1.40 when the
immersion medium is special oil with a refractive index
of 1.51. Other common immersion media are water and
glycerin. Objectives designed for special immersion
media usually have a color-coded ring inscribed around
the circumference of the objective barrel as listed in Table
3 and described below.
· Color Codes: Many microscope manufacturers label
their objectives with color codes to help in rapid
identification of the magnification. The dark blue color
code on the objective illustrated in Figure 9 indicates the
linear magnification is 60x. This is very helpful when
you have a nosepiece turret containing 5 or 6 objectives
and you must quickly select a specific magnification.
Some specialized objectives have an additional color code
that indicates the type of immersion medium necessary
to achieve the optimum numerical aperture. Immersion
lenses intended for use with oil have a black color ring,
while those intended for use with glycerin have an orange
ring. Objectives designed to image living organisms in
aqueous media are designated water immersion objectives
Table 3
Color-Coded Rings on Microscope Objectives
Immersion color code
a
Immersion type
Black
Orange
White
Red
Magnification color code
Black
Brown
Red
Yellow
Green
Turquoise blue
Light blue
Cobalt (dark) blue
White (cream)
Oil immersion
Glycerol immersion
Water immersion
Special
b
Magnification
1x, 1.25x
2x, 2.5x
4x, 5x
10x
16x, 20x
25x, 32x
40x, 50x
60x, 63x
100x
a
Narrow colored ring located near the specimen end of
objective.
b
Narrow band located closer to the mounting thread than the
immersion code.
with a white ring and highly specialized objectives for
unusual immersion media often are engraved with a red
ring. Table 3 lists current magnification and imaging
media color codes in use by most manufacturers.
· Specialized Optical Properties: Microscope
objectives often have design parameters that optimize
performance under certain conditions. For example, there
are special objectives designed for polarized illumination
(signified by the abbreviations P, Po, Pol, or SF, and/or
having all barrel engravings painted red), phase contrast
(PH, and/or green barrel engravings), differential
interference contrast (DIC), and many other abbreviations
for additional applications. The apochromat objective
illustrated in Figure 9 is optimized for DIC
photomicrography and this is indicated on the barrel. The
capital H beside the DIC marking indicates that the
objective must be used with a specific DIC prism
optimized for high-magnification applications.
There are some applications that do not require
objectives designed to be corrected for cover glass
thickness. These include objectives used to observe
uncovered specimens in reflected light metallurgical
specimens, integrated circuit inspection, micro
machinery, biological smears, and other applications that
require observation of uncovered objects. Other common
abbreviations found on microscope objective barrels,
which are useful in identifying specific properties, are
listed in Table 4 (17).
12
OPTICAL MICROSCOPY
Table 4
Davidson and Abramowitz
Specialized Objective Designations
Abbreviation
Type
Phase, PHACO, PC, Ph 1,2, 3, etc.
DL, DM, PLL, PL, PM, PH, NL, NM, NH
Phase contrast, using phase condenser annulus 1, 2, 3, etc.
Phase contrast: dark low, dark medium, positive low, positive low, positive
medium, positive high contrast (regions with higher refractive index appear
darker); negative low, negative medium, negative high contrast (regions with
higher refractive index appear lighter)
Strain-free, low birefringence, for polarized light
UV transmitting (down to approx. 340 nm), for UV-excited epifluorescence
Metallographic (no coverslip)
No coverslip
Surface illumination (specimen illuminated through objective lens), as contrasted to
dia- or transillumination
Transmitted light
For use in bright or dark field (hell, dunkel)
Dark field
Designed primarily for heating stage
Designed to be used with universal stage (magnification/NA applied for use
with glass hemisphere; divine both values by 1.51 when hemisphere is not used)
Interferometry; noncontact; multiple beam (Tolanski)
P, Po, Pol, SF
U, UV, Universal
M
NC, NCG
EPI
TL
BBD, HD, B\D
D
H
U, UT
DI; MI; TI Michelson
a
Many of the designation codes are manufacturer specific.
light waves passing through the periphery of a lens are
not brought into identical focus with those passing closer
to the center. Waves passing near the center of the lens
are refracted only slightly, whereas waves passing near
the periphery are refracted to a greater degree resulting
in the production of different focal points along the optical
axis. This is one of the most serious resolution artifacts
because the image of the specimen is spread out rather
than being in sharp focus. Spherical aberrations are very
important in terms of the resolution of the lens because
they affect the coincident imaging of points along the
optical axis and degrade the performance of the lens,
which will seriously affect specimen sharpness and clarity.
These lens defects can be reduced by limiting the outer
edges of the lens from exposure to light using diaphragms
and also by utilizing aspherical lens surfaces within the
system. The highest-quality modern microscope
objectives address spherical aberrations in a number of
ways including special lens-grinding techniques,
additional lens elements of different curvatures, improved
glass formulations, and better control of optical pathways.
Chromatic Aberration: This type of optical defect
is a result of the fact that white light is composed of
numerous wavelengths. When white light passes through
a convex lens, the component wavelengths are refracted
according to their frequency. Blue light is refracted to
the greatest extent followed by green and red light, a
phenomenon commonly referred to as dispersion. The
inability of the lens to bring all of the colors into a
Optical Aberrations
Lens errors or aberrations in optical microscopy are
caused by artifacts arising from the interaction of light
with glass lenses (2-5, 19-24). There are two primary
causes of aberration: (i) geometrical or spherical
aberrations are related to the spherical nature of the lens
and approximations used to obtain the Gaussian lens
equation; and (ii) chromatic aberrations that arise from
variations in the refractive indices of the wide range of
frequencies found in visible light.
In general, the effects of optical aberrations are to
induce faults in the features of an image being observed
through a microscope. These artifacts were first addressed
in the eighteenth century when physicist John Dollond
discovered that chromatic aberration would be reduced
or corrected by using a combination of two different types
of glass (flint and crown) in the fabrication of lenses (13).
Later, during the nineteenth century, achromatic objectives
with high numerical aperture were developed, although
there were still geometrical problems with the lenses.
Modern glass formulations coupled with advanced
grinding and manufacturing techniques have all but
eliminated most aberrations from today’s microscope
objectives, although careful attention must still be paid
to these effects, especially when conducting quantitative
high-magnification
video
microscopy
and
photomicrography (23).
Spherical Aberration: These artifacts occur when
13
OPTICAL MICROSCOPY
common focus results in a slightly different image size
and focal point for each predominant wavelength group.
This leads to color fringes surrounding the image.
Lens corrections were first attempted in the latter part
of the 18th century when Dollond, Lister and others
devised ways to reduce longitudinal chromatic aberration
(13). By combining crown glass and flint glass (each type
has a different dispersion of refractive index), they
succeeded in bringing the blue rays and the red rays to a
common focus, near but not identical with the green rays.
This combination is termed a lens doublet where each lens
has a different refractive index and dispersive properties.
Lens doublets are also known as achromatic lenses or
achromats for short, derived from the Greek terms a
meaning without and chroma meaning color. This simple
form of correction allows the image points at 486
nanometers in the blue region and 656 nanometers in the
red region to now coincide. This is the most widely used
objective lens and is commonly found on laboratory
microscopes. Objectives that do not carry a special
inscription stating otherwise are likely to be achromats.
Achromats are satisfactory objectives for routine
laboratory use, but because they are not corrected for all
colors, a colorless specimen detail is likely to show, in
white light, a pale green color at best focus (the so-called
secondary spectrum).
A proper combination of lens thickness, curvature,
refractive index, and dispersion allows the doublet to
reduce chromatic aberration by bringing two of the
wavelength groups into a common focal plane. If fluorspar
is introduced into the glass formulation used to fabricate
the lens, then the three colors red, green, and blue can be
brought into a single focal point resulting in a negligible
amount of chromatic aberration (23). These lenses are
known as apochromatic lenses and they are used to build
very high-quality chromatic aberration-free microscope
objectives. Modern microscopes utilize this concept and
today it is common to find optical lens triplets made with
three lens elements cemented together, especially in
higher-quality objectives. For chromatic aberration
correction, a typical 10x achromat microscope objective
is built with two lens doublets (Figure 8(a)). Apochromat
objectives usually contain two lens doublets and a lens
triplet (Figure 8(c)) for advanced correction of both
chromatic and spherical aberrations.
Despite longitudinal (or axial) chromatic aberration
correction, apochromat objectives also exhibit another
chromatic defect. Even when all three main colors are
brought to identical focal planes axially, the point images
of details near the periphery of the field of view, are not
the same size; e.g., the blue image of a detail is slightly
larger than the green image or the red image in white light,
thus causing color ringing of specimen details at the outer
Davidson and Abramowitz
regions of the field of view (23). This defect is known as
lateral chromatic aberration or chromatic difference of
magnification. It is the compensating eyepiece, with
chromatic difference of magnification just the opposite
of that of the objective, which is utilized to correct for
lateral chromatic aberration. Because this defect is also
found in higher magnification achromats, compensating
eyepieces are frequently used for such objectives, too.
Indeed, many manufacturers design their achromats with
a standard lateral chromatic error and use compensating
eyepieces for all their objectives. Such eyepieces often
carry the inscription K or C or Compens. As a result,
compensating eyepieces have build-in lateral chromatic
error and are not, in themselves, perfectly corrected.
Coverslip Correction: It is possible for the user to
inadvertently introduce spherical aberration into a wellcorrected system (2, 23). For example, when using high
magnification and high numerical aperture dry objectives
(NA = 0.85-0.95), the correct thickness of the cover glass
(suggested 0.17 mm) is critical; hence the inclusion of a
correction collar on such objectives to enable adjustment
for incorrect cover glass thickness. Similarly, the
insertion of accessories in the light path of finite tube
length objectives may introduce aberrations, apparent
when the specimen is refocused, unless such accessories
have been properly designed with additional optics. Figure
10 illustrates how internal lenses operate in an objective
designed for coverslip correction.
Other Geometrical Aberrations: These include a
variety of effects including astigmatism, field curvature,
and comatic aberrations, which are corrected with proper
lens fabrication.
Curvature of field in the image is an aberration that is
familiar to most experienced microscopists. This artifact
is the natural result of using lenses that have curved
surfaces. When visible light is focused through a curved
lens, the image plane produced by the lens will be curved.
When the image is viewed in the eyepieces (oculars) of a
microscope, it either appears sharp and crisp in the center
or on the edges of the viewfield but not both. Normally,
this is not a serious problem when the microscopist is
routinely scanning samples to observe their various
features. It is a simple matter to use the fine focus knob
to correct small deficiencies in specimen focus.
However, for photomicrography, field curvature can be a
serious problem, especially when a portion of the
photomicrograph is out of focus.
Modern microscopes deal with field curvature by
correcting this aberration using specially designed flatfield objectives. These specially corrected objectives
have been named plan or plano and are the most common
type of objective in use today. Plan objectives are also
corrected for other optical artifacts such as spherical and
14
OPTICAL MICROSCOPY
Davidson and Abramowitz
objects and are most severe when the microscope is out
of alignment (23). In this instance, the image of a point
is asymmetrical, resulting in a comet-like (hence, the term
coma) shape. The comet shape may have its tail pointing
toward the center of the field of view or away depending
upon whether the comatic aberration has a positive or
negative value. Coma may occur near the axial area of
the light path, and/or the more peripheral area. These
aberrations are usually corrected along with spherical
aberrations by designing lens elements of various shapes
to eliminate this error. Objectives that are designed to
yield excellent images for wide field of view eyepieces,
have to be corrected for coma and astigmatism using a
specially-designed multi-element optic in the tube lens
to avoid these artifacts at the periphery of the field of
view.
Astigmatism aberrations are similar to comatic
aberrations, however these artifacts depend more strongly
on the obliquity of the light beam (23). This defect is
found at the outer portions of the field of view of
uncorrected lenses. The off-axis image of a specimen
point appears as a line instead of a point. What is more,
depending on the angle of the off-axis rays entering the
lens, the line image may be oriented in either of two
different directions, tangentially or radially. Astigmatism
errors are usually corrected by design of the objectives
to provide precise spacing of individual lens elements as
well as appropriate lens shapes and indices of refraction.
The correction of astigmatism is often accomplished in
conjunction with the correction of field curvature
aberrations.
chromatic aberrations. In the case of a plan objective that
also has been mostly corrected for chromatic aberration,
the objective is referred to as a plan achromat. This is
also the case for fluorite and apochromatic objectives,
which have the modified names: plan fluorite and plan
apochromat.
Adding field curvature lens corrections to an objective
that has already been corrected for optical aberrations can
often add a significant number of lens elements to the
objective. For example, the typical achromat objective
has two lens doublets and a hemispherical lens, making
of total of five lens elements. In contrast, a comparable
plan achromat objective has three doublets and three single
lenses for a total of nine lens elements, making it
considerably more difficult to fabricate. As we have seen,
the number of lens elements increases as lenses are
corrected for spherical errors as well as chromatic and
field curvature aberrations. Unfortunately, as the number
of lens elements increases so does the cost of the
objective.
Sophisticated plan apochromatic objectives that are
corrected for spherical, chromatic, and field curvature
aberrations can contain as many as eighteen to twenty
separate lens elements, making these objectives the most
expensive and difficult to manufacture. Plan apochromatic
objectives can cost upward of $3,000 to $5,000 each for
high-magnification units that also have a high numerical
aperture. For most photomicrography applications,
however, it is not absolutely necessary to have the best
correction, although this is heavily dependent upon the
purpose, the specimen, and the desired magnification
range. When cost is important (when isn’t it?), it is often
wise to select more modestly priced plan fluorite
objectives that have a high degree of correction,
especially the more modern versions. These objectives
provide crisp and sharp images with minimal field
curvature, and will be sufficient for most
photomicrography applications.
Field curvature is very seldom totally eliminated, but
it is often difficult to detect edge curvature with most
plan-corrected objectives and it does not show up in
photomicrographs (19, 23). This artifact is more severe
at low magnifications and can be a problem with stereo
microscopes. Manufacturers have struggled for years to
eliminate field curvature in the large objectives found in
stereo microscopes. In the past ten years, companies like
Leica, Nikon, Olympus, and Zeiss, have made great strides
in the quality of optics used to build stereo microscopes
and, while the artifacts and aberrations have not been
totally eliminated, high-end models are now capable of
producing superb photomicrographs.
Comatic aberrations are similar to spherical
aberrations, but they are mainly encountered with off-axis
Eyepieces (Oculars)
Eyepieces work in combination with microscope
objectives to further magnify the intermediate image so
that specimen details can be observed. Ocular is an
alternative name for eyepieces that has been widely used
in the literature, but to maintain consistency during this
discussion we will refer to all oculars as eyepieces. Best
results in microscopy require that objectives be used in
combination with eyepieces that are appropriate to the
correction and type of objective. Inscriptions on the side
of the eyepiece describe its particular characteristics and
function.
The eyepiece illustrated in Figure 11 is inscribed with
UW (not illustrated), which is an abbreviation for the ultra
wide viewfield. Often eyepieces will also have an H
designation, depending upon the manufacturer, to indicate
a high-eyepoint focal point that allows microscopists to
wear glasses while viewing samples. Other common
inscriptions often found on eyepieces include WF for
wide field; UWF for ultra wide field; SW and SWF for
15
OPTICAL MICROSCOPY
super wide field; HE for high eyepoint; and CF for
eyepieces intended for use with CF corrected objectives
(2, 3, 5, 19, 23, 24). As discussed above, compensating
eyepieces are often inscribed with K, C, Comp, or
Compens, as well as the magnification. Eyepieces used
with flat-field objectives are sometimes labeled PlanComp. Magnification factors of common eyepieces
range from 5x to 25x, and usually contain an inscription,
such as A/24, which indicates the field number is 24, in
reference to the diameter (in millimeters) of the fixed
diaphragm in the eyepiece. Many eyepieces also have a
focus adjustment and a thumbscrew that allows their
position to be fixed. Manufactures now often produce
eyepieces having rubber eye-cups that serve both to
position the eyes the proper distance from the front lens,
and to block room light from reflecting off the lens
surface and interfering with the view.
Davidson and Abramowitz
way between these lenses there is a fixed circular opening
or internal diaphragm which, by its size, defines the
circular field of view that is observed in looking into the
microscope. The simplest kind of negative eyepiece, or
Huygenian eyepiece, is found on most routine
microscopes fitted with achromatic objectives. Although
the Huygenian eye and field lenses are not well corrected,
their aberrations tend to cancel each other out. More
highly corrected negative eyepieces have two or three lens
elements cemented and combined together to make the
eye lens. If an unknown eyepiece carries only the
magnification inscribed on the housing, it is most likely
to be a Huygenian eyepiece, best suited for use with
achromatic objectives of 5x-40x magnification.
The other main type of eyepiece is the positive
eyepiece with a diaphragm below its lenses, commonly
known as the Ramsden eyepiece. This eyepiece has an
eye lens and field lens that are also plano-convex, but the
field lens is mounted with the curved surface facing
towards the eye lens. The front focal plane of this
eyepiece lies just below the field lens, at the level of the
eyepiece fixed diaphragm, making this eyepiece readily
adaptable for mounting graticules. To provide better
correction, the two lenses of the Ramsden eyepiece may
be cemented together.
Simple eyepieces such as the Huygenian and Ramsden
and their achromatized counterparts will not correct for
residual chromatic difference of magnification in the
intermediate image, especially when used in combination
with high magnification achromatic objectives as well as
fluorite or apochromatic objectives. To remedy this in
finite microscopy systems, manufacturers produce
compensating eyepieces that introduce an equal, but
opposite, chromatic error in the lens elements.
Compensating eyepieces may be either of the positive or
negative type, and must be used at all magnifications with
fluorite, apochromatic and all variations of plan objectives
(they can also be used to advantage with achromatic
objectives of 40x and higher).
In recent years, modern microscope objectives have
their correction for chromatic difference of magnification
either built into the objectives themselves (Olympus and
Nikon) or corrected in the tube lens (Leica and Zeiss),
thus eliminating the need for compensation correction
of the eyepieces.
Compensating eyepieces play a crucial role in helping
to eliminate residual lateral chromatic aberrations
inherent in the design of highly corrected objectives.
Hence, it is preferable that the microscopist uses the
compensating eyepieces designed by a particular
manufacturer to accompany that manufacturer’s highercorrected objectives. Use of an incorrect eyepiece with
an apochromatic objective designed for a finite (160 or
Figure 11. Cutaway diagram of a typical periplan eyepiece. The
fixed aperture diaphragm is positioned between lens group 1 and
lens group 2, where the intermediate image is formed. The eyepiece
has a protective eyecup that makes viewing the specimen more
comfortable for the microscopist.
There are two major types of eyepieces that are
grouped according to lens and diaphragm arrangement:
the negative eyepieces with an internal diaphragm
between the lenses, and positive eyepieces that have a
diaphragm below the lenses of the eyepiece. Negative
eyepieces have two lenses: the upper lens, which is closest
to the observer’s eye, is called the eye-lens and the lower
lens (beneath the diaphragm) is often termed the field lens.
In their simplest form, both lenses are plano-convex, with
convex sides facing the specimen. Approximately mid16
OPTICAL MICROSCOPY
170 millimeter) tube length microscope results in
dramatically increased contrast with red fringes on the
outer diameters and blue fringes on the inner diameters
of specimen detail. Additional problems arise from a
limited flatness of the viewfield in simple eyepieces, even
those corrected with eye-lens doublets.
More advanced eyepiece designs resulted in the
Periplan eyepiece (Figure 11), which contains seven lens
elements that are cemented into a doublet, a triplet, and
two individual lenses. Design improvements in periplan
eyepieces lead to better correction for residual lateral
chromatic aberration, increased flatness of field, and a
general overall better performance when used with higher
power objectives.
Modern microscopes feature vastly improved plancorrected objectives in which the primary image has much
less curvature of field than older objectives. In addition,
most microscopes now feature much wider body tubes
that have accommodated greatly increased the size of
intermediate images. To address these new features,
manufacturers now produce wide-eyefield eyepieces that
increase the viewable area of the specimen by as much as
40 percent. Because the strategies of eyepiece-objective
correction techniques vary from manufacturer to
manufacturer, it is very important (as stated above) to use
only eyepieces recommended by a specific manufacturer
for use with their objectives.
Our recommendation is to carefully choose the
objective first, then purchase an eyepiece that is designed
to work in conjunction with the objective. When choosing
eyepieces, it is relatively easy to differentiate between
simple and more highly compensating eyepieces. Simple
eyepieces such as the Ramsden and Huygenian (and their
more highly corrected counterparts) will appear to have a
blue ring around the edge of the eyepiece diaphragm when
viewed through the microscope or held up to a light
source. In contrast, more highly corrected compensating
eyepieces with have a yellow-red-orange ring around the
diaphragm under the same circumstances. Modern noncompensating eyepieces are fully corrected and show no
color. Most of the modern microscopes have all
corrections done in the objectives themselves or have a
final correction in the tube lens. Such microscopes do
not need compensating eyepieces.
The properties of several common commercially
available eyepieces are listed according to type in Table
5 (19, 23). The three major types of eyepieces listed in
Table 5 are finder, wide field, and super wide field. The
terminology used by various manufacturers can be very
confusing and careful attention should be paid to their
sales brochures and microscope manuals to ensure that
the correct eyepieces are being used with a specific
objective. In Table 5, the abbreviations that designate wide
Davidson and Abramowitz
field and super widefield eyepieces are coupled to their
design for high eyepoint, and are WH and SWH,
respectively. The magnifications are either 10x or 15x
and the Field Numbers range from 14 to 26.5, depending
upon the application. The diopter adjustment is
approximately the same for all eyepieces and many also
contain either a photomask or micrometer graticule.
Light rays emanating from the eyepiece intersect at
the exit pupil or eyepoint (Ramsden disc) where the front
of the microscopist’s eye should be placed in order to
see the entire field of view (usually 8-10 mm above the
eye lens). By increasing the magnification of the eyepiece,
the eyepoint is drawn closer to the upper surface of the
eye lens, making it much more difficult for microscopists
to use, especially if he or she wears eyeglasses. Specially
designed high eyepoint eyepieces have been manufactured
that feature eyepoint viewing distances approaching 2025 mm above the surface of the eye lens. These improved
eyepieces have larger diameter eye lenses that contain
more optical elements and usually feature improved
flatness of field. Such eyepieces are often designated
with the inscription “H” somewhere on the eyepiece
housing, either alone or in combination with other
abbreviations, as discussed above. We should mention
that high-eyepoint eyepieces are especially useful for
microscopists who wear eyeglasses to correct for near
or far sightedness, but they do not correct for several other
visual defects, such as astigmatism. Today, high eyepoint
eyepieces are very popular, even with people who do not
wear eyeglasses, because the large eye clearance reduces
fatigue and makes viewing images through the microscope
much more comfortable.
At one time, eyepieces were available in a wide range
of magnifications extending from 6.3x to 30x and
sometimes even higher for special applications. These
eyepieces are very useful for observation and
photomicrography with low-power objectives.
Unfortunately, with higher power objectives, the problem
of empty magnification (magnification without increased
clarity) becomes important when using very high
magnification eyepieces and these should be avoided.
Today most manufacturers restrict their eyepiece
offerings to those in the 10x to 20x ranges. The diameter
of the viewfield in an eyepiece is expressed as a field of
view number or field number (FN), as discussed above.
Information about the field number of an eyepiece can
yield the real diameter of the object viewfield using the
formula (23):
Viewfield Diameter = FN / (MO x MT) (2)
Where FN is the field number in millimeters, MO is the
magnification of the objective, and MT is the tube lens
17
OPTICAL MICROSCOPY
Table 5
Properties of Commercial Eyepieces
Eyepiece Type
Finder Eyepieces
Descriptive
Abbreviation
PSWH
10x
PWH
10x
Field Number
26.5
22
Diopter
Adjustment
-8 ~ +2
-8 ~ +2
Super Wide Field
Eyepieces
Diameter of
Micrometer
Graticule
—
—
SWH
10x H
CROSSWH
10x H
WH
15x
WH
10x H
26.5
26.5
22
14
22
-8 ~ +2
-8 ~ +2
-8 ~ +2
-8 ~ +2
-8 ~ +2
diopter
correction
diopter
correction
crossline
—
—
—
magnification factor (if any). Applying this formula to
the super wide field eyepiece listed in Table 5, we arrive
at the following for a 40x objective with a tube lens
magnification of 1.25:
Table 1 lists the viewfield diameters over the common
range of objectives that would occur using this eyepiece.
Table 6 Range of Useful Magnification
(500-1000 x NA of Objective)
Eyepieces
15x
20x
10x
12.5x
—
—
—
x
x
—
—
x
x
x
—
x
x
x
x
x
x
x
x
—
x
x
x
—
—
x
x
x
—
—
x
x
—
—
—
diopter
correction
24
24
specimen detail without adding unnecessary artifacts. For
instance, to achieve a magnification of 250x, the
microscopist could choose a 25x eyepiece coupled to a
10x objective. An alternative choice for the same
magnification would be a 10x eyepiece with a 25x
objective. Because the 25x objective has a higher
numerical aperture (approximately 0.65) than does the 10x
objective (approximately 0.25), and considering that
numerical aperture values define an objective’s resolving
power, it is clear that the latter choice would be the best.
If photomicrographs of the same viewfield were made
with each objective/eyepiece combination described
above, it would be obvious that the 10x eyepiece/25x
objective duo would produce photomicrographs that
excelled in specimen detail and clarity when compared
to the alternative combination.
Numerical aperture of the objective/condenser system
defines the range of useful magnification for an
objective/eyepiece combination (19, 22-24). There is a
minimum magnification necessary for the detail present
in an image to be resolved, and this value is usually rather
arbitrarily set as 500 times the numerical aperture (500 x
NA). At the other end of the spectrum, the maximum
useful magnification of an image is usually set at 1000
times the numerical aperture (1000 x NA).
Magnifications higher than this value will yield no further
useful information or finer resolution of image detail,
and will usually lead to image degradation. Exceeding
the limit of useful magnification causes the image to suffer
from the phenomenon of empty magnification (19),
where increasing magnification through the eyepiece or
intermediate tube lens only causes the image to become
Viewfield Diameter = 26.5 / 40 x 1.25 = 0.53 mm (3)
Objective
(NA)
Wide Field Eyepieces
35 SWH
10x
31/4 x “41/4” 31/4 x “41/4” 35mm
photo
photo
photo
mask
mask
mask
Remarks
2.5x
(0.08)
4x
(0.12)
10x
(0.35)
25x
(0.55)
40x
(0.70)
60x
(0.95)
100x
(1.42)
Davidson and Abramowitz
25x
Care should be taken in choosing eyepiece/objective
combinations to ensure the optimal magnification of
18
OPTICAL MICROSCOPY
more magnified with no corresponding increase in detail
resolution. Table 6 lists the common objective/eyepiece
combinations that fall into the range of useful
magnification.
Eyepieces can be adapted for measurement purposes
by adding a small circular disk-shaped glass graticule at
the plane of the fixed aperture diaphragm of the eyepiece.
Graticules usually have markings, such as a measuring rule
or grid, etched onto the surface. Because the graticule
lies in the same conjugate plane as the fixed eyepiece
diaphragm, it appears in sharp focus superimposed on the
image of the specimen. Eyepieces using graticules usually
contain a focusing mechanism (helical screw or slider)
that allows the image of the graticule to be brought into
focus. A stage micrometer is needed to calibrate the
eyepiece scale for each objective.
Davidson and Abramowitz
focus to be adjusted for proper illumination of the
specimen. Correct positioning of the condenser with
relation to the cone of illumination and focus on the
specimen is critical to quantitative microscopy and
optimum photomicrography. Care must be taken to
guarantee that the condenser aperture is opened to the
correct position with respect to objective numerical
aperture. When the aperture is opened too much, stray
light generated by refraction of oblique light rays from
the specimen can cause glare and lower the overall
contrast. On the other hand, when the aperture is closed
too far, the illumination cone is insufficient to provide
adequate resolution and the image is distorted due to
refraction and diffraction from the specimen.
Condenser Systems
The substage condenser gathers light from the
microscope light source and concentrates it into a cone
of light that illuminates the specimen with parallel beams
of uniform intensity from all azimuths over the entire
viewfield. It is critical that the condenser light cone be
properly adjusted to optimize the intensity and angle of
light entering the objective front lens. Each time an
objective is changed, a corresponding adjustment must
be performed on the substage condenser aperture iris
diaphragm to provide the proper light cone for the
numerical aperture of the new objective.
A simple two-lens Abbe condenser is illustrated in
Figure 12. In this figure, light from the microscope
illumination source passes through the aperture or
condenser diaphragm, located at the base of the condenser,
and is concentrated by internal lens elements, which then
project light through the specimen in parallel bundles
from every azimuth. The size and numerical aperture of
the light cone is determined by adjustment of the aperture
diaphragm. After passing through the specimen (on the
microscope slide), the light diverges to an inverted cone
with the proper angle (2q in Figure 12) to fill the front
lens of the objective (2, 18-24).
Aperture adjustment and proper focusing of the
condenser are of critical importance in realizing the full
potential of the objective. Specifically, appropriate use
of the adjustable aperture iris diaphragm (incorporated
into the condenser or just below it) is most important in
securing correct illumination, contrast, and depth of field.
The opening and closing of this iris diaphragm controls
the angle of illuminating rays (and thus the aperture) which
pass through the condenser, through the specimen and then
into the objective. Condenser height is controlled by a
rack and pinion gear system that allows the condenser
Figure 12. Condenser/objective configuration for optical
microscopy. An Abbe two-lens condenser is illustrated showing
ray traces through the optical train of the microscope. The aperture
diaphragm restricts light entering the condenser before it is refracted
by the condenser lens system into the specimen. Immersion oil is
used in the contact beneath the θunderside of the slide and the
condenser top lens, and also between the objective and cover slip.
The objective angular aperture ( ) controls the amount of light
entering the objective.
The simplest and least corrected (also the least
expensive) condenser is the Abbe condenser that, in its
simplest form, has two optical lens elements which
produce an image of the illuminated field diaphragm that
19
OPTICAL MICROSCOPY
is not sharp and is surrounded by blue and red color at the
edges. As a result of little optical correction, the Abbe
condenser is suited mainly for routine observation with
objectives of modest numerical aperture and
magnification. The primary advantages of the Abbe
condenser are the wide cone of illumination that the
condenser is capable of producing as well as its ability to
work with long working distance objectives. The
manufacturers supply most microscopes with an Abbe
condenser as the default and these condensers are real
workhorses for routine laboratory use.
Table 7
aberration (green wavelengths) but not for chromatic
aberration. These condensers feature five lens elements
and are capable of focusing light in a single plane.
Aplanatic condensers are capable of producing excellent
black and white photomicrographs when used with green
light generated by either a laser source or by use of an
interference filter with tungsten-halide illumination.
The highest level of correction for optical aberration
is incorporated in the aplanatic-achromatic condenser (2,
5). This condenser is well corrected for both chromatic
and spherical aberrations and is the condenser of choice
for use in critical color photomicrography with white
light. A typical aplanatic-achromatic condenser features
eight internal lens elements cemented into two doublets
and four single lenses.
Engravings found on the condenser housing include
its type (achromatic, aplanatic, etc.), the numerical
aperture, and a graduated scale that indicates the
approximate adjustment (size) of the aperture diaphragm.
As we mentioned above, condensers with numerical
apertures above 0.95 perform best when a drop of oil is
applied to their upper lens in contact with the undersurface
of the specimen slide. This ensures that oblique light rays
emanating from the condenser are not reflected from
underneath the slide, but are directed into the specimen
without deviation. In practice, this can become tedious
and is not commonly done in routine microscopy, but is
essential when working at high resolutions and for
accurate photomicrography using high-power (and
numerical aperture) objectives.
Another important consideration is the thickness of
the microscope slide, which is as crucial to the condenser
as coverslip thickness is to the objective. Most
commercial producers offer slides that range in thickness
between 0.95 and 1.20 mm with the most common being
very close to 1.0 mm. A microscope slide of thickness
1.20 mm is too thick to be used with most high numerical
aperture condensers that tend to have a very short working
distance. While this does not greatly matter for routine
specimen observation, the results can be devastating with
precision photomicrography. We recommend that
microscope slides be chosen that have a thickness of 1.0
± 0.05 mm, and that they be thoroughly cleaned prior to
use.
When the objective is changed, for example from a
10x to 20x, the aperture diaphragm of the condenser must
also be adjusted to provide a light cone that matches the
numerical aperture of the new objective. There is a small
painted arrow or index mark located on this knurled knob
or lever that indicates the relative size of the aperture when
compared to the linear gradation on the condenser
housing. Many manufacturers will synchronize this
gradation to correspond to the approximate numerical
Condenser Aberration Corrections
Condenser
Type
Abbe
Aplanatic
Achromatic
Aplanaticachromatic
Aberrations
Corrected
Spherical
Chromatic
—
x
—
x
—
—
x
x
Davidson and Abramowitz
The next highest level of condenser correction is split
between the aplanatic and achromatic condensers that are
corrected exclusively for either spherical (aplanatic) or
chromatic (achromatic) optical aberrations.
Achromatic condensers typically contain four lens
elements and have a numerical aperture of 0.95, the
highest attainable without requiring immersion oil (5).
This condenser is useful for both routine and critical
laboratory analysis with dry objectives and also for black
and white or color photomicrography.
A critical factor in choosing substage condensers is
the numerical aperture performance, which will be
necessary to provide an illumination cone adequate for
the objectives. The condenser numerical aperture
capability should be equal to or slightly less than that of
the highest objective numerical aperture. Therefore, if
the largest magnification objective is an oil-immersion
objective with a numerical aperture of 1.40, then the
substage condenser should also have an equivalent
numerical aperture to maintain the highest system
resolution. In this case, immersion oil would have to be
applied between the condenser top lens in contact with
the underside of the microscope slide to achieve the
intended numerical aperture (1.40) and resolution. Failure
to use oil will restrict the highest numerical aperture of
the system to 1.0, the highest obtainable with air as the
imaging medium (2, 5, 17-24).
Aplanatic condensers are well corrected for spherical
20
OPTICAL MICROSCOPY
of illumination cones with a single condenser (18).
In practice, this problem can be solved in several ways.
For low power objectives (below 10x), it may be
necessary to unscrew the top lens of the condenser in order
to fill the field of view with light. Other condensers are
produced with a flip-top upper lens to accomplish this
more readily. Some manufacturers now produce a
condenser that flips over completely when used with low
power objectives. Other companies incorporate auxiliary
correction lenses in the light path for securing proper
illumination with objectives less than 10x, or produce
special low-power and low-numerical aperture
condensers. When the condenser is used without its top
lens, the aperture iris diaphragm is opened wide and the
field diaphragm, now visible at the back of the objective,
aperture of the condenser. For example, if the
microscopist has selected a 10x objective of numerical
aperture 0.25, then the arrow would be placed next the
value 0.18-0.20 (about 80 percent of the objective
numerical aperture) on the scale inscribed on the
condenser housing.
Often, it is not practical to use a single condenser with
an entire range of objectives (2x to 100x) due to the broad
range of light cones that must be produced to match
objective numerical apertures. With low-power
objectives in the range 2x to 5x, the illumination cone
will have a diameter between 6-10 mm, while the highpower objectives (60x to 100x) need a highly focused
light cone only about 0.2-0.4 mm in diameter. With a
fixed focal length, it is difficult to achieve this wide range
Table 8
Davidson and Abramowitz
Substage Condenser Applications
Condenser
Type
Brightfield
Achromat /
Aplanat
N.A. 1.3
•
[10x~100x]
Achromat
Swing-out
N.A. 0.90
•
[4x~100x]
Low-Power
N.A. 0.20
•
[1x~10x]
Darkfield
Phase
Contrast
Phase
Contrast Abbe
N.A. 1.25
•
•
[up to N.A. 0.65]
•
[10x~100x]
Phase
Contrast
Achromat
N.A. 0.85
•
•
[up to N.A. 0.70]
•
[4x~100x]
DIC Universal
Achromat /
Aplanat
•
•
[up to N.A. 0.70]
•
[10x~100x]
Darkfield, dry
N.A. 0.80~0.95
•
[4x~40x]
Darkfield, oil
N.A. 1.20~1.43
•
[4x~100x]
Strain-Free
Achromat
Swing-out
N.A. 0.90
•
DIC
Polarizing
•
[10x, 20x, 40x, 100x]
•
[4x~100x]
21
OPTICAL MICROSCOPY
Table 9 Depth of Field and Image Depth
Magnification
4x
10x
20x
40x
60x
100x
a
Davidson and Abramowitz
a
Numerical Aperture
Depth of Field (M)
0.10
0.25
0.40
0.65
0.85
0.95
15.5
8.5
5.8
1.0
0.40
0.19
Image Depth (mm)
0.13
0.80
3.8
12.8
29.8
80.0
Source: Nikon
serves as if it were the aperture diaphragm. Flip-top
condensers are manufactured in a variety of configurations
with numerical apertures ranging from 0.65 to 1.40.
Those condensers having a numerical aperture value of
0.95 or less are intended for use with dry objectives. Fliptop condensers that have a numerical aperture greater than
0.95 are intended for use with oil-immersion objectives
and they must have a drop of oil placed between the
underside of the microscope slide and the condenser top
lens when examining critical samples.
In addition to the common brightfield condensers
discussed above, there are a wide variety of specialized
models suited to many different applications. Substage
condensers have a great deal of interchangeability among
different applications. For instance, the DIC universal
achromat/aplanat condenser is useful for brightfield,
darkfield, and phase contrast, in addition to the primary
DIC application. Other condensers have similar
interchangeability.
space. This interchange of terms can lead to confusion,
especially when the terms are both used specifically in
terms of depth of field.
The geometric image plane might be expected to
represent an infinitely thin section of the specimen, but
even in the absence of aberrations, each image point is
spread into a diffraction figure that extends above and
below this plane (2, 4, 17). The Airy disk, discussed in the
section on Image Formation, represents a section
through the center of the image plane. This increases the
effective in-focus depth of the Z-axis Airy disk intensity
profile that passes through slightly different specimen
planes.
Depth of focus varies with numerical aperture and
magnification of the objective, and under some conditions,
high numerical aperture systems (usually with higher
magnification power) have deeper focus depths than do
those systems of low numerical aperture, even though the
depth of field is less (4). This is particularly important in
photomicrography because the film emulsion or digital
camera sensor must be exposed at a plane that is in focus.
Small errors made to focus at high magnification are not
as critical as those made with very low magnification
objectives. Table 9 presents calculated variations in the
depth of field and image depth in the intermediate image
plane in a series of objectives with increasing numerical
aperture and magnification.
Depth of Field and Depth of Focus
When considering resolution in optical microscopy,
a majority of the emphasis is placed on point-to-point
resolution in the plane perpendicular to the optical axis.
Another important aspect to resolution is the axial
resolving power of an objective, which is measured
parallel to the optical axis and is most often referred to
as depth of field (2, 4, 5, 22). Axial resolution, like
horizontal resolution, is determined by the numerical
aperture of the objective only, with the eyepiece merely
magnifying the details resolved and projected in the
intermediate image plane.
Just as in classical photography, depth of field is
determined by the distance from the nearest object plane
in focus to that of the farthest plane also simultaneously
in focus. In microscopy depth of field is very short and
usually measured in terms of microns. The term depth
of focus, which refers to image space, is often used
interchangeably with depth of field, which refers to object
Reflected Light Microscopy
Reflected light microscopy is often referred to as
incident light, epi-illumination, or metallurgical
microscopy, and is the method of choice for fluorescence
and for imaging specimens that remain opaque even when
ground to a thickness of 30 microns (25). The range of
specimens falling into this category is enormous and
includes most metals, ores, ceramics, many polymers,
semiconductors (unprocessed silicon, wafers, and
integrated circuits), slag, coal, plastics, paint, paper, wood,
leather, glass inclusions, and a wide variety of specialized
22
OPTICAL MICROSCOPY
Davidson and Abramowitz
materials (25-28). Because light is unable to pass through adjustment knobs raise or lower the stage in large or small
these specimens, it must be directed onto the surface and increments to bring the specimen into sharp focus.
eventually returned to the microscope objective by either
Another variation of the upright reflected light
specular or diffused reflection. As mentioned above, such microscope is the inverted microscope—of the Le
illumination is most often referred to as episcopic Chatelier design (25). On the inverted stand, the specimen
illumination, epi-illumination, or vertical illumination is placed on the stage with its surface of interest facing
(essentially originating from above), in contrast to downward. The primary advantage of this design is that
diascopic (transmitted) illumination that passes through samples can be easily examined when they are far too large
a specimen. Today, many microscope manufacturers offer to fit into the confines of an upright microscope. Also,
models that permit the user to alternate or simultaneously the only the side facing the objectives need be perfectly
conduct investigations using vertical and transmitted flat. The objectives are mounted on a nosepiece under
illumination. Reflected light microscopy is frequently the stage with their front lenses facing upward towards
the domain of industrial microscopy, especially in the the specimen and focusing is accomplished either by
rapidly growing semiconductor arena, and thus represents moving the nosepiece or the entire stage up and down.
a most important segment of microscopical studies.
Inverted microscope stands incorporate the vertical
A typical upright compound
reflected light (illustrated in
Figure 13) microscope also
equipped for transmitted light has
two eyepiece viewing tubes and
often a trinocular tube head for
mounting a conventional or digital/
video camera system. Standard
equipment eyepieces are usually
of 10x magnification, and most
microscopes are equipped with a
nosepiece capable of holding four
to six objectives. The stage is
mechanically controlled with a
specimen holder that can be
translated in the X- and Ydirections and the entire stage unit
is capable of precise up and down
movement with a coarse and fine
focusing mechanism. Built-in
light sources range from 20 and
100 watt tungsten-halide bulbs to
higher energy mercury vapor or
xenon lamps that are used in
fluorescence microscopy. Light
passes from the lamp house
through a vertical illuminator
interposed above the nosepiece Figure 13. Components of a modern microscope configured for both transmitted and
but below the underside of the reflected light. This cutaway diagram reveals the ray traces and lens components of the
viewing tube head. The speci- microscope’s optical trains. Also illustrated are the basic microscope components including
men’s top surface is upright two lamp houses, the microscope built-in vertical and base illuminators, condenser, objectives,
(usually without a cover slip) on eyepieces, filters, sliders, collector lenses, field, and aperture diaphragms.
the stage facing the objective,
which has been rotated into the microscope’s optical axis. illuminator into the body of the microscope. Many types
The vertical illuminator is horizontally oriented at a 90- of objectives can be used with inverted reflected light
degree angle to the optical axis of the microscope and microscopes, and all modes of reflected light illumination
parallel to the table top, with the lamp housing attached may be possible: brightfield, darkfield, polarized light,
to the back of the illuminator. The coarse and fine differential interference contrast, and fluorescence. Many
23
OPTICAL MICROSCOPY
Davidson and Abramowitz
described as universal illuminators because, with several
additional accessories and little or no dismantling, the
microscope can easily be switched from one mode of
reflected light microscopy to another. Often, reflectors
can be removed from the light path altogether in order to
perform transmitted light observation. Such universal
illuminators may include a partially reflecting plane glass
surface (the half-mirror) for brightfield, and a fully
silvered reflecting surface with an elliptical, centrally
located clear opening for darkfield observation. The bestdesigned vertical illuminators include condensing lenses
to gather and control the light, an aperture iris diaphragm
and a pre-focused, centerable iris diaphragm to permit the
desirable Köhler illumination (2, 25).
The vertical illuminator should also make provision
for the insertion of filters for contrast and
photomicrography, polarizers, analyzers, and compensator
plates for polarized light and differential interference
contrast illumination. In vertical illuminators designed
for with infinity-corrected objectives, the illuminator may
also include a body tube lens. Affixed to the back end of
the vertical illuminator is a lamphouse, which usually
contains a tungsten-halide lamp. For fluorescence work,
the lamphouse can be replaced with one containing a
mercury burner. The lamp may be powered by the
electronics built into the microscope stand, or in
fluorescence, by means of an external transformer or
power supply.
In reflected light microscopy, absorption and
diffraction of the incident light rays by the specimen often
lead to readily discernible variations in the image, from
black through various shades of gray, or color if the
specimen is colored. Such specimens are known as
amplitude specimens and may not require special contrast
methods or treatment to make their details visible. Other
specimens show so little difference in intensity and/or
color that their feature details are extremely difficult to
discern and distinguish in brightfield reflected light
microscopy. Such specimens behave much like the phase
specimens so familiar in transmitted light work. Such
objects require special treatment or contrast methods that
will be described in the next section.
of the inverted microscopes have built-in 35 millimeter
and/or large format cameras or are modular to allow such
accessories to be attached. Some of the instruments
include a magnification changer for zooming in on the
image, contrast filters, and a variety of reticules. Because
an inverted microscope is a favorite instrument for
metallographers, it is often referred to as a metallograph
(25, 27). Manufacturers are largely migrating to using
infinity-corrected optics in reflected light microscopes,
but there are still thousands of fixed tube length
microscopes in use with objectives corrected for a tube
length between 160 and 210 millimeters.
In the vertical illuminator, light travels from the light
source, usually a 12 volt 50 or 100 watt tungsten halogen
lamp, passes through collector lenses, through the variable
aperture iris diaphragm opening and through the opening
of a variable and centerable pre-focused field iris
diaphragm. The light then strikes a partially silvered plane
glass reflector (Figure 13), or strikes a fully silvered
periphery of a mirror with elliptical opening for darkfield
illumination. The plane glass reflector is partially silvered
on the glass side facing the light source and anti-reflection
coated on the glass side facing the observation tube in
brightfield reflected illumination. Light is thus deflected
downward into the objective. The mirrors are tilted at an
angle of 45 degrees to the path of the light travelling along
the vertical illuminator.
The light reaches the specimen, which may absorb
some of the light and reflect some of the light, either in a
specular or diffuse manner. Light that is returned upward
can be captured by the objective in accordance with the
objective’s numerical aperture and then passes through
the partially silvered mirror. In the case of infinitycorrected objectives, the light emerges from the objective
in parallel (from every azimuth) rays projecting an image
of the specimen to infinity (25). The parallel rays enter
the body tube lens, which forms the specimen image at
the plane of the fixed diaphragm opening in the eyepiece
(intermediate image plane). It is important to note, that
in these reflected light systems, the objective serves a
dual function: on the way down as a matching wellcorrected condenser properly aligned; on the way up as
an image-forming objective in the customary role of an
objective projecting the image-carrying rays toward the
eyepiece.
Optimal performance is achieved in reflected light
illumination when the instrument is adjusted to produce
Köhler illumination. A function of Köhler illumination
(aside from providing evenly dispersed illumination) is
to ensure that the objective will be able to deliver excellent
resolution and good contrast even if the source of light is
a coil filament lamp.
Some modern reflected light illuminators are
Contrast Enhancing Techniques
Some specimens are considered amplitude objects
because they absorb light partially or completely, and can
thus be readily observed using conventional brightfield
microscopy. Others that are naturally colored or
artificially stained with chemical color dyes can also be
seen. These stains or natural colors absorb some part of
the white light passing through and transmit or reflect other
colors. Often, stains are combined to yield contrasting
24
OPTICAL MICROSCOPY
colors, e.g. blue haemotoxylin stain for cell nuclei
combined with pink eosin for cytoplasm. It is a common
practice to utilize stains on specimens that do not readily
absorb light, thus rendering such objects visible to the
eye.
Contrast produced by the absorption of light,
brightness, or color has been the classical means of
imaging specimens in brightfield microscopy. The ability
of a detail to stand out against the background or other
adjacent details is a measure of specimen contrast. In
terms of a simple formula, contrast can be described as
(18):
Percent Contrast = ((BI – SI) x 100)/BI
Davidson and Abramowitz
mounted in a variety of contrast-enhancing mediums such
as air or the commercial medium Styrax. The difference
in refractive indices improves the contrast of these
colorless objects and renders their outlines and markings
more visible. The following sections describe many of
the more complex techniques used by present-day
microscopists to improve specimen contrast.
Darkfield Microscopy
Darkfield illumination requires blocking out of the
central light rays that ordinarily pass through or around
the specimen and allowing only oblique rays to illuminate
the specimen. This method is a simple and popular method
for imaging unstained specimens, which appear as brightly
illuminated objects on a dark background. Oblique light
rays emanating from a darkfield condenser strike the
specimen from every azimuth and are diffracted, reflected,
and refracted into the microscope objective (5, 18, 19,
28). This technique is illustrated in Figure 14. If no
specimen is present and the numerical aperture of the
condenser is greater than that of the objective, the oblique
rays cross and all such rays will miss entering the objective
because of the obliquity. The field of view will appear
dark.
(4)
Where BI is the intensity of the background and SI is the
specimen intensity. From this equation, it is evident that
specimen contrast refers to the relationship between the
highest and lowest intensity in the image.
For many specimens in microscopy, especially
unstained or living material, contrast is so poor that the
object remains essentially invisible regardless of the
ability of the objective to resolve or clearly separate
details. Often, for just such specimens, it is important
not to alter them by killing or treatment with chemical
dyes or fixatives. This necessity has led microscopists
to experiment with contrast-enhancing techniques for over
a hundred years in an attempt to improve specimen
visibility and to bring more detail to the image. It is a
common practice to reduce the condenser aperture
diaphragm below the recommended size or to lower the
substage condenser to increase specimen contrast.
Unfortunately, while these maneuvers will indeed increase
contrast, they also seriously reduce resolution and
sharpness.
An early and currently used method of increasing
contrast of stained specimens utilizes color contrast
filters, gelatin squares (from Kodak), or interference
filters in the light path (18, 19). For example, if a
specimen is stained with a red stain, a green filter will
darken the red areas thus increasing contrast. On the other
hand, a green filter will lighten any green stained area.
Color filters are very valuable aids to specimen contrast,
especially when black and white photomicrography is the
goal. Green filters are particularly valuable for use with
achromats, which are spherically corrected for green
light, and phase contrast objectives, which are designed
for manipulation of wavelength assuming the use of green
light, because phase specimens are usually transparent and
lack inherent color. Another simple technique for contrast
improvement involves the selection of a mounting
medium with a refractive index substantially different
from that of the specimen. For example, diatoms can be
Figure 14. Schematic configuration for darkfield microscopy.
The central opaque light stop is positioned beneath the condenser
to eliminate zeroth order illumination of the specimen. The
condenser produces a hollow cone of illumination that strikes the
specimen at oblique angles. Some of the reflected, refracted, and
diffracted light from the specimen enters the objective front lens.
25
OPTICAL MICROSCOPY
Davidson and Abramowitz
In terms of Fourier
use a condenser designed
optics, darkfield illumiespecially for darkfield,
nation removes the zeroth
i.e. to transmit only
order (unscattered light)
oblique rays (28, 29).
from the diffraction pattern
There are several varieties
formed at the rear focal plane
including dry darkfield
of the objective (5).
condensers with air
Oblique rays, now diffracted
between the top of the
by the specimen and yielding
condenser
and
the
1st, 2nd, and higher
underside of the slide.
diffracted orders at the rear
Immersion
darkfield
focal plane of the objective,
condensers are also
proceed onto the image
available. These require
plane where they interfere
the use of a drop of
with one another to produce
immersion oil (some are
an image of the specimen.
designed to use water
This results in an image
instead)
establishing
formed exclusively from
contact between the top of
higher order diffraction
the condenser and the
intensities scattered by the
underside of the specimen
specimen. Specimens that Figure 15. Darkfield photomicrograph of the diatom slide. The immersion
have smooth reflective Arachnoidiscus ehrenbergi taken at high magnification using oil darkfield condenser has
surfaces produce images immersion optics and a 100x objective.
internal mirrored surfaces
due, in part, to reflection of
and passes rays of great
light into the objective (18, 19). In situations where the obliquity and free of chromatic aberration, producing the
refractive index is different from the surrounding medium best results and blackest background.
or where refractive index gradients occur (as in the edge
Darkfield objects are quite spectacular to see and
of a membrane), light is refracted by the specimen. Both objects of very low contrast in brightfield shine brilliantly
instances of reflection and refraction produce relatively in darkfield. Such illumination is best for revealing
small angular changes in the direction of light allowing outlines, edges, and boundaries. A high magnification
some to enter the objective. In contrast, some light darkfield image of a diatom is illustrated in Figure 15.
striking the specimen is also diffracted, producing a 180degree arc of light that passes through the entire numerical
Rheinberg Illumination
aperture range of the objective. The resolving power of
the objective is the same in darkfield illumination as found
Rheinberg illumination, a form of optical staining, was
under brightfield conditions, but the optical character is initially demonstrated by the British microscopist Julius
the image is not as faithfully reproduced.
Rheinberg to the Royal Microscopical Society and the
There are several pieces of equipment that are utilized Quekett Club (England) nearly a hundred years ago (18,
to produce darkfield illumination. The simplest is a spider 28). This technique is a striking variation of low to
stop (Figure 14) placed just under the bottom lens (in the medium power darkfield illumination using colored
front focal plane) of the substage condenser (5, 18, 19, gelatin or glass filters to provide rich color to both the
28). Both the aperture and field diaphragms are opened specimen and background. The central opaque darkfield
wide to pass oblique rays. The central opaque stop (you stop is replaced with a transparent, colored, circular stop
can make one by mounting a coin on a clear glass disk) inserted into a transparent ring of a contrasting color
blocks out the central rays. This device works fairly well, (illustrated in Figure 16). These stops are placed under
even with the Abbe condenser, with the 10x objective up the bottom lens of the condenser. The result is a specimen
to 40x or higher objectives having a numerical aperture rendered in the color of the ring with a background having
no higher than 0.65. The diameter of the opaque stop the color of the central spot. An example of
should be approximately 16-18 millimeters for a 10x photomicrography using Rheinberg illumination is
objective of numerical aperture 0.25 to approximately 20- illustrated in Figure 17 with the spiracle and trachea of a
24 millimeters for 20x and 40x objectives of numerical silkworm larva.
apertures approaching 0.65.
For more precise work and blacker backgrounds,
26
OPTICAL MICROSCOPY
Davidson and Abramowitz
index or thickness (or both). This diffracted light, lagging
behind by approximately 1/4 wavelength, arrives at the
image plane out of step (also termed out of phase) with
the undeviated light but, in interference, essentially
undiminished in intensity. The result is that the image at
the eyepiece level is so lacking in contrast as to make the
details almost invisible.
Zernike succeeded in devising a method—now known
as Phase Contrast microscopy—for making unstained,
phase objects yield contrast images as if they were
amplitude objects. Amplitude objects show excellent
contrast when the diffracted and direct light are out of
step (display a phase difference) by 1/2 of a wavelength
(18). Zernike’s method was to speed up the direct light
by 1/4 wavelength so that the difference in wavelength
between the direct and deviated light for a phase specimen
would now be 1/2 wavelength. As a result, the direct and
diffracted light arriving at the image level of the eyepiece
would be able to produce destructive interference (see
the section on image formation for absorbing objects
previously described). Such a procedure results in the
details of the image appearing darker against a lighter
background. This is called dark or positive phase contrast.
A schematic illustration of the basic phase contrast
microscope configuration is illustrated in Figure 18.
Another possible course, much less often used, is to
arrange to slow down the direct light by 1/4 wavelength
so that the diffracted light and the direct light arrive at the
eyepiece in step and can interfere constructively (2, 5,
18). This arrangement results in a bright image of the
details of the specimen on a darker background, and is
called negative or bright contrast.
Phase contrast involves the separation of the direct
zeroth order light from the diffracted light at the rear focal
plane of the objective. To do this, a ring annulus is placed
in position directly under the lower lens of the condenser
at the front focal plane of the condenser, conjugate to the
objective rear focal plane. As the hollow cone of light
from the annulus passes through the specimen undeviated,
it arrives at the rear focal plane of the objective in the
shape of a ring of light. The fainter light diffracted by the
specimen is spread over the rear focal plane of the
objective. If this combination were allowed, as is, to
proceed to the image plane of the eyepiece, the diffracted
light would be approximately 1/4 wavelength behind the
direct light. At the image plane, the phase of the diffracted
light would be out of phase with the direct light, but the
amplitude of their interference would be almost the same
as that of the direct light (5, 18). This would result in
very little specimen contrast.
To speed up the direct undeviated zeroth order light, a
phase plate is installed with a ring shaped phase shifter
attached to it at the rear focal plane of the objective. The
Figure 16. Schematic microscope configuration for Rheinberg
illumination. Light passes through the central/annular filter pack
prior to entering the condenser. Zeroth order light from the central
filter pervades the specimen and background and illuminates it
with higher order light from the annular filter. The filter colors in
this diagram are a green central and red annular.
Phase Contrast Microscopy
Research by Frits Zernike during the early 1930s
uncovered phase and amplitude differences between
zeroth order and deviated light that can be altered to
produce favorable conditions for interference and
contrast enhancement (30, 31). Unstained specimens that
do not absorb light are called phase objects because they
slightly alter the phase of the light diffracted by the
specimen, usually by retarding such light approximately
1/4 wavelength as compared to the undeviated direct light
passing through or around the specimen unaffected.
Unfortunately, our eyes as well as camera film are unable
to detect these phase differences. To reiterate, the human
eye is sensitive only to the colors of the visible spectrum
or to differing levels of light intensity (related to wave
amplitude).
In phase specimens, the direct zeroth order light passes
through or around the specimen undeviated. However, the
light diffracted by the specimen is not reduced in
amplitude as it is in a light-absorbing object, but is slowed
by the specimen because of the specimen’s refractive
27
OPTICAL MICROSCOPY
Davidson and Abramowitz
because achromats are spherically corrected for green
light.
The accessories needed for phase contrast work are a
substage phase contrast condenser equipped with annuli
and a set of phase contrast objectives, each of which has a
phase plate installed. The condenser usually has a
brightfield position with an aperture diaphragm and a
rotating turret of annuli (each phase objective of different
magnification requires an annulus of increasing diameter
as the magnification of the objective increases). Each
phase objective has a darkened ring on its back lens. Such
objectives can also be used for ordinary brightfield
transmitted light work with only a slight reduction in
image quality. A photomicrograph of a hair cross sections
from a fetal mouse taken using phase contrast illumination
is illustrated in Figure 19.
Figure 17. Spiracle and trachea from silkworm larva photographed
at low magnification under Rheinberg illumination using a blue
central and yellow annulus filters and a 2x objective.
narrow area of the phase ring is optically thinner than the
rest of the plate. As a result, undeviated light passing
through the phase ring travels a shorter distance in
traversing the glass of the objective than does the
diffracted light. Now, when the direct undeviated light
and the diffracted light proceed to the image plane, they
are 1/2 wavelength out of phase with each other. The
diffracted and direct light can now interfere destructively
so that the details of the specimen appear dark against a
lighter background (just as they do for an absorbing or
amplitude specimen). This is a description of what takes
place in positive or dark phase contrast.
If the ring phase shifter area of the phase plate is made
optically thicker than the rest of the plate, direct light is
slowed by 1/4 wavelength. In this case, the zeroth order
light arrives at the image plane in step (or in phase) with
the diffracted light, and constructive interference takes
place. The image appears bright on a darker background.
This type of phase contrast is described as negative or
bright contrast (2, 5, 18, 19).
Because undeviated light of the zeroth order is much
brighter than the faint diffracted light, a thin absorptive
transparent metallic layer is deposited on the phase ring
to bring the direct and diffracted light into better balance
of intensity to increase contrast. Also, because speeding
up or slowing down of the direct light is calculated on a
1/4 wavelength of green light, the phase image will appear
best when a green filter is placed in the light path (a green
interference filter is preferable). Such a green filter also
helps achromatic objectives produce their best images,
Figure 18. Schematic configuration for phase contrast microscopy.
Light passing through the phase ring is first concentrated onto the
specimen by the condenser. Undeviated light enters the objective
and is advancedd by the phase plate before interference at the
rear focal plane of the objective.
28
OPTICAL MICROSCOPY
Davidson and Abramowitz
anisotropic crystals (32-34).
When anisotropic crystals refract light, the resulting
rays are polarized and travel at different velocities. One
of the rays travels with the same velocity in every direction
through the crystal and is termed the ordinary ray. The
other ray travels with a velocity that is dependent upon
the propagation direction within the crystal. This light
ray is termed the extraordinary ray. The retardation
between the ordinary and extraordinary ray increases with
increasing crystal thickness. The two independent
refractive indices of anisotropic crystals are quantified
in terms of their birefringence, a measure of the
difference in refractive index. Thus, the birefringence
(B) of a crystal is defined as:
Polarized Light
Many transparent solids are optically isotropic,
meaning that the index of refraction is equal in all
directions throughout the crystalline lattice. Examples of
isotropic solids are glass, table salt (sodium chloride),
many polymers, and a wide variety of both organic and
inorganic compounds (32, 33).
B = |nhigh – nlow|
(5)
Where nhigh is the largest refractive index and nlow is the
smallest. This expression holds true for any part or
fragment of an anisotropic crystal with the exception of
light waves propagated along the optical axis of the crystal.
As mentioned above, light that is doubly refracted through
anisotropic crystals is polarized with the vibration
directions of the polarized ordinary and extraordinary light
waves being oriented perpendicular to each other. We
can now examine how anisotropic crystals behave under
polarized illumination in a polarizing microscope.
A polarizer place beneath the substage condenser is
oriented such that polarized light exiting the polarizer is
plane polarized in a vibration direction that is east-west
with respect to the optic axis of the microscope stand.
Polarized light enters the anisotropic crystal where it is
refracted and divided into two separate components
vibrating parallel to the crystallographic axes and
perpendicular to each other. The polarized light waves
then pass through the specimen and objective before
reaching a second polarizer (usually termed the analyzer)
that is oriented to pass a polarized vibration direction
perpendicular to that of the substage polarizer. Therefore,
the analyzer passes only those components of the light
waves that are parallel to the polarization direction of the
analyzer. The retardation of one ray with respect to
another is caused the difference in speed between the
ordinary and extraordinary rays refracted by the
anisotropic crystal (33, 34). A schematic illustration of
microscope configuration for crossed polarized
illumination is presented in Figure 20.
Now we will consider the phase relationship and
velocity differences between the ordinary and
extraordinary rays after they pass through a birefringent
crystal. These rays are oriented so that they are vibrating
at right angles to each other. Each ray will encounter a
slightly different electrical environment (refractive index)
Figure 19. Photomicrograph of hair cross sections from a fetal
mouse taken using phase contrast optics and a 20x objective.
Crystals are classified as being either isotropic or
anisotropic depending upon their optical behavior and
whether or not their crystallographic axes are equivalent.
All isotropic crystals have equivalent axes that interact
with light in a similar manner, regardless of the crystal
orientation with respect to incident light waves. Light
entering an isotropic crystal is refracted at a constant
angle and passes through the crystal at a single velocity
without being polarized by interaction with the electronic
components of the crystalline lattice.
Anisotropic crystals, on the other hand, have
crystallographically distinct axes and interact with light
in a manner that is dependent upon the orientation of the
crystalline lattice with respect to the incident light. When
light enters the optical axis of anisotropic crystals, it acts
in a manner similar to interaction with isotropic crystals
and passes through at a single velocity. However, when
light enters a non-equivalent axis, it is refracted into two
rays each polarized with their vibration directions oriented
at right angles to one another, and traveling at different
velocities. This phenomenon is termed double or birefraction and is seen to a greater or lesser degree in all
29
OPTICAL MICROSCOPY
Davidson and Abramowitz
Where Γ is the quantitative retardation of the material, t
is the thickness of the birefringent crystal (or material)
and B is birefringence as defined above (33). Factors
contributing to the value of retardation are the magnitude
of the difference in refractive indices for the
environments seen by the ordinary and extraordinary rays
and also the sample thickness. Obviously, the greater the
difference in either refractive indices or thickness, the
greater the degree of retardation. Early observations made
on the mineral calcite indicated that thicker calcite
crystals caused greater differences in splitting of the
images seen through the crystals. This agrees with the
equation above that states retardation will increase with
crystal (or sample) thickness.
When the ordinary and extraordinary rays emerge from
the birefringent crystal, they are still vibrating at right
angles with respect to one another. However, the
component vectors of these waves that pass through the
analyzer are vibrating in the same plane. Because one
wave is retarded with respect to the other, interference
(either constructive or destructive) occurs between the
waves as they pass through the analyzer. The net result is
that some birefringent samples (in white light) acquire a
spectrum of color when observed through crossed
polarizers.
Polarized light microscopy requires strain-free
objectives and condensers to avoid depolarization effects
on the transmitted light (32-34). Most polarized
Figure 20. Schematic microscope configuration for observing
birefringent specimens under crossed polarized illumination. White
light passing through the polarizer is plane polarized and
concentrated onto the birefringent specimen by the condenser.
Light rays emerging from the specimen interfere when they are
recombined in the analyzer, subtracting some of the wavelengths
of white light, thus producing a myriad of tones and colors.
as it enters the crystal and this will affect the velocity at
which ray passes through the crystal (32, 33). Because
of the difference in refractive indices, one ray will pass
through the crystal at a slower rate than the other ray. In
other words, the velocity of the slower ray will be retarded
with respect to the faster ray. This retardation can be
quantified using the following equation:
Retardation (G) = thickness (t) x Birefringence (B)
or
Γ = t x |nhigh - nlow|
Figure 21. Photomicrograph of high-density columnar-hexatic
liquid crystalline calf thymus DNA at a concentration of
approximately 450 milligrams/milliliter. This concentration of DNA
is approaching that found in sperm heads, virus capsids, and
dinoflagellate chromosomes. The image was recorded using a
polarized light microscope and the 10x objective.
(6)
(7)
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OPTICAL MICROSCOPY
Davidson and Abramowitz
microscopes are equipped with a centerable stage that has
free 360-degree rotation about the optical axis of the
microscope. A Bertrand lens is commonly inserted into
the light path so that conoscopic images of birefringence
patterns can be observed at the back of the objective.
Manufacturers also offer a wide range of compensators
and light retarders (full-wave and quarter-wave plates) for
quantitative birefringence measurements and for adding
color to polarized light photomicrographs. Birefringent
DNA liquid crystals photographed using crossed polarized
illumination are illustrated in Figure 21.
Hoffman Modulation Contrast
The Hoffman Modulation Contrast system is designed
to increase visibility and contrast in unstained and living
material by detecting optical gradients (or slopes) and
converting them into variations of light intensity. This
ingenious technique was invented by Dr. Robert Hoffman
in 1975, and employs several accessories that have been
adapted to the major commercial microscopes (16, 18).
A schematic illustration of microscope configuration for
Hoffman modulation contrast is presented in Figure 22.
An optical amplitude spatial filter, termed a modulator
by Hoffman, is inserted at the rear focal plane of an
achromat or planachromat objective (although higher
correction can also be used). Light intensity passing
through this system varies above and below an average
value, which by definition, is then said to be modulated.
Objectives useful for modulation contrast can cover the
entire magnification range of 10x to 100x. The modulator
has three zones: a small, dark zone near the periphery of
the rear focal plane which transmits only one percent of
light; a narrow gray zone which transmits 15 percent; and
the remaining clear or transparent zone, covering most
of the area at the back of the objective, which transmits
100 percent of the light (5, 16, 18). Unlike the phase
plate in phase contrast microscopy, the Hoffman
modulator is designed not to alter the phase of light
passing through any of the zones. When viewed under
modulation contrast optics, transparent objects that are
essentially invisible in ordinary brightfield microscopy
take on an apparent three-dimensional appearance dictated
by phase gradients. The modulator does not introduce
changes in the phase relationship of light passing through
the system, but influences the principal zeroth order
maxima. Higher order diffraction maxima are unaffected.
Measurements using a Michelson interferometer confirm
that the spatial coherency of light passed through a
Hoffman-style modulator varies (if any) by a factor of
less than l/20 (5).
Below the stage, a condenser with rotating turret is
utilized to hold the remaining components of the Hoffman
Figure 22. Schematic illustration of microscope configuration
for Hoffman modulation contrast. Light passing through the
polarizer and slit is concentrated onto the specimen by the
condenser. After passing through the specimen light enters the
objective and is filtered by the modulator plate in the rear focal
plane of the objective.
Modulation Contrast system. The turret condenser has a
brightfield opening with an aperture iris diaphragm for
regular brightfield microscopy and for alignment and
establishing proper conditions of Köhler illumination for
the microscope. At each of the other turret openings,
there is an off-center slit that is partially covered with a
small rectangular polarizer. The size of the slit/polarizer
combination is different for each objective of different
magnification; hence the need for a turret arrangement.
When light passes through the off-axis slit, it is imaged
at the rear focal plane of the objective (also termed the
Fourier plane) where the modulator has been installed.
Like the central annulus and phase ring in phase contrast
microscopy, the front focal plane of the condenser
31
OPTICAL MICROSCOPY
Davidson and Abramowitz
major microscope manufacturers now offer modulation
contrast objectives in fluorite-correction grades, and
apochromats can be obtained by special order. Older
objectives can often be retrofitted with a modulator made
by Modulation Optics, Inc., the company founded by Dr.
Robert Hoffman specifically to build aftermarket and
custom systems.
containing the off-axis slit plate is optically conjugate to
the modulator in objective rear focal plane. Image
intensity is proportional to the first derivative of the
optical density in the specimen, and is controlled by the
zeroth order of the phase gradient diffraction pattern.
Below the condenser, a circular polarizer is placed on
the light exit port of the microscope (note that both
polarizers are below the specimen). The rotation of this
polarizer can control the effective width of the slit
opening. For example, a crossing of both polarizers at
90 degrees to each other results in narrowing the slit so
that its image falls within the gray area of the modulator
(16, 18). The part of the slit controlled by the polarizer
registers on the bright area of the modulator. As the
polarizer is rotated, contrast can be varied for best effect.
A very narrow slit produces images that are very high in
contrast with a high degree of coherence. Optical section
imaging is also optimized when the slit is adjusted to its
narrowest position. When the circular polarizer is
oriented with its vibration direction parallel to that of the
polarizer in the slit, the effective slit width is at a
maximum. This reduces overall image contrast and
coherence, but yields much better images of thicker
objects where large differences in refractive index exist.
In modern advanced modulation contrast systems, both
the modulator and the slit are offset from the optical axis
of the microscope (18). This arrangement permits fuller
use of the numerical aperture of the objective and results
in good resolution of specimen detail. Shapes and details
are rendered in shadowed, pseudo three-dimensional
appearance. These appear brighter on one side, gray in
the central portion, and darker on the other side, against a
gray background. The modulator converts optical phase
gradients in details (steepness, slope, rate of change in
refractive index, or thickness) into changes in the intensity
of various areas of the image at the plane of the eyepiece
diaphragm. Resulting images have an apparent threedimensional appearance with directional sensitivity to
optical gradients. The contrast (related to variations in
intensity) of the dark and bright areas against the gray gives
a shadowed pseudo-relief effect. This is typical of
modulation contrast imaging. Rotation of the polarizer
alters the contrast achieved and the orientation of the
specimen on the stage (with respect to the polarizer and
offset slit) may dramatically improve or degrade contrast.
There are numerous advantages as well as limitations
to modulation contrast. Some of the advantages include
fuller use of the numerical aperture of the objective
yielding excellent resolution of details along with good
specimen contrast and visibility. Although many standard
modulation contrast objectives are achromats or
planachromats, it is also possible to use objectives with a
higher degree of correction for optical aberration. Many
Figure 23. Duck-billed dinosaur bone thin section photographed
using a combination of Hoffman modulation contrast and polarized
light illumination using a 20x objective. Note the pseudo threedimensional relief evident throughout the photomicrograph as a
result of amplitude gradients (Hoffman modulation contrast). The
vivid colors are due to interference of white light at the analyzer
(polarized light).
In addition to the advantages of using higher numerical
apertures with modulation contrast, it is also possible to
do optical sectioning with this technique (16, 18).
Sectioning allows the microscopist to focus on a single
thin plane of the specimen without interference from
confusing images arising in areas above or below the plane
that is being focused on. The depth of a specimen is
measured in a direction parallel to the optical axis of the
microscope. Focusing the image establishes the correct
specimen-to-image distance, allowing interference of the
diffracted waves to occur at a pre-determined plane (the
image plane) positioned at a fixed distance from the
eyepiece. This enables diffracting objects that occur at
different depth levels in the specimen to be viewed
separately, provided there is sufficient contrast. The entire
depth of a specimen can be optically sectioned by
sequentially focusing on each succeeding plane. In this
system, depth of field is defined as the distance from one
level to the next where imaging of distinct detail occurs,
32
OPTICAL MICROSCOPY
Davidson and Abramowitz
direction perpendicular to the propagation direction of
the light beam, enters the beam-splitting modified
Wollaston prism and is split into two rays, vibrating
perpendicular to each other (Figure 24). These two rays
travel close together but in slightly different directions.
The rays intersect at the front focal plane of the condenser,
where they pass traveling parallel and extremely close
together with a slight path difference, but they are vibrating
perpendicular to each other and are therefore unable to
cause interference. The distance between the rays, called
the shear, is so minute that it is less than the resolving
ability of the objective (5, 18, 36).
The split beams enter and pass through the specimen
where their wave paths are altered in accordance with the
specimen’s varying thickness, slopes, and refractive
and is controlled by the numerical aperture of the objective
(5, 16, 18). Higher numerical aperture objectives exhibit
very shallow depths of field and the opposite holds for
objectives of lower numerical aperture. The overall
capability of an objective to isolate and focus on a specific
optical section diminishes as the optical homogeneity of
the specimen decreases.
Birefringent objects (rock thin sections, crystals,
bone, etc.), that can confuse images in DIC, can be
examined because the specimen is not situated between
the two polarizers (18). Further, specimens can be
contained in plastic or glass vessels without deterioration
of the image due to polarization effects, because such
vessels are also above both polarizers, not between them.
This allows the Hoffman system to be far more useful
than DIC in the examination and photomicrography of cell,
tissue, and organ culture performed in plastic containers.
Hoffman modulation contrast can be simultaneously
combined with polarized light microscopy to achieve
spectacular effects in photomicrography. Figure 23
illustrates a dinosaur bone thin section photographed with
a combination of Hoffman modulation contrast and plane
polarized illumination. Note the pseudo three-dimensional
relief apparent throughout the micrograph and the beautiful
coloration provided by polarized light.
Differential Interference Contrast
In the mid 1950s a French optics theoretician named
Georges Nomarski improved the method for detecting
optical gradients in specimens and converting them into
intensity differences (15). Today there are several
implementations of this design, which are collectively
called differential interference contrast (DIC). Living or
stained specimens, which often yield poor images when
viewed in brightfield illumination, are made clearly visible
by optical rather than chemical means (2, 4, 5, 15, 1822).
In transmitted light DIC, light from the lamp is passed
through a polarizer located beneath the substage
condenser, in a manner similar to polarized light
microscopy. Next in the light path (but still beneath the
condenser) is a modified Wollaston prism that splits the
entering beam of polarized light into two beams traveling
in slightly different direction (illustrated in Figure 24).
The prism is composed of two halves cemented together
(36). Emerging light rays vibrate at 90 degrees relative
to each other with a slight path difference. A different
prism is needed for each objective of different
magnification. A revolving turret on the condenser allows
the microscopist to rotate the appropriate prism into the
light path when changing magnifications.
The plane polarized light, vibrating only in one
Figure 24. Schematic illustration of microscope configuration for
differential interference contrast. Light is polarized in a single
vibration plane by the polarizer before entering the lower modified
Wollaston prism that acts as a beam splitter. Next, the light passes
through the condenser and sample before the image is reconstructed
by the objective. Above the objective, a second Wollaston
(Nomarski) prism acts as a beam-combiner and passes the light to
the analyzer, where it interferes both constructively and destructively.
33
OPTICAL MICROSCOPY
(a)
Davidson and Abramowitz
(b)
Figure 25 (a) Nomarski transmitted light differential interference contrast (DIC) photomicrograph of mouthparts from a blowfly.
(b) Reflected light differential interference contrast photomicrograph illustration defects on the surface of a ferro-silicon alloy. Both
images were captured using the 10x objective and a first-order retardation plate.
indices. These variations cause alterations in the wave
path of both beams passing through areas of the specimen
details lying close together. When the parallel beams
enter the objective, they are focused above the rear focal
plane where they enter a second modified Wollaston
prism that combines the two beams. This removes the
shear and the original path difference between the beam
pairs. As a result of having traversed the specimen, the
paths of the parallel beams are not of the same length
(optical path difference) for differing areas of the
specimen.
In order for the beams to interfere, the vibrations of
the beams of different path length must be brought into
the same plane and axis. This is accomplished by placing
a second polarizer (analyzer) above the upper Wollaston
beam-combining prism. The light then proceeds toward
the eyepiece where it can be observed as differences in
intensity and color. The design results in one side of a
detail appearing bright (or possibly in color) while the
other side appears darker (or another color). This shadow
effect bestows a pseudo three-dimensional appearance to
the specimen (18).
In some microscopes, the upper modified Wollaston
prism is combined in a single fitting with the analyzer
incorporated above it. The upper prism may also be
arranged so it can be moved horizontally. This allows for
varying optical path differences by moving the prism,
providing the user a mechanism to alter the brightness
and color of the background and specimen. Because of
the prism design and placements, the background will be
homogeneous for whatever color has been selected.
The color and/or light intensity effects shown in the
image are related especially to the rate of change in
refractive index, specimen thickness, or both. Orientation
of the specimen can have pronounced effect on the relieflike appearance and often, rotation of the specimen by
180 degrees changes a hill into a valley or visa versa. The
three-dimensional appearance is not representing the true
geometric nature of the specimen, but is an exaggeration
based on optical thickness. It is not suitable for accurate
measurement of actual heights and depths.
There are numerous advantages in DIC microscopy as
compared particular to phase and Hoffman modulation
contrast microscopy. With DIC, it is also possible to make
fuller use of the numerical aperture of the system and to
provide optical staining (color). DIC also allows the
microscope to achieve excellent resolution. Use of full
objective aperture enables the microscopist to focus on
a thin plane section of a thick specimen without confusing
images from above or below the plane. Annoying halos,
often encountered in phase contrast, are absent in DIC
images, and common achromat and fluorite objectives can
be used for this work. A downside is that plastic tissue
culture vessels and other birefringent specimens yield
confusing images in DIC. Also, high-quality apochromatic
objectives are now designed to be suitable for DIC. Figure
24 presents transmitted and reflected light DIC
photomicrographs of the mouthparts of a blowfly
34
OPTICAL MICROSCOPY
(transmitted DIC) and surface defects in a ferro-silicate
alloy (reflected DIC). Both photomicrographs were made
using a retardation plate with a 10x objective.
Davidson and Abramowitz
mode of microscopy in which the specimen, subsequent
to excitation, gives off its own light (6). The emitted light
re-radiates spherically in all directions, regardless of the
direction of the exciting light. A schematic diagram of
the configuration for fluorescence microscopy is
presented in Figure 26.
Fluorescence microscopy has advantages based upon
attributes not as readily available in other optical
microscopy techniques. The use of fluorochromes has
made it possible to identify cells and sub-microscopic
cellular components and entities with a high degree of
specificity amidst non-fluorescing material. What is
more, the fluorescence microscope can reveal the
presence of fluorescing material with exquisite
sensitivity. An extremely small number of fluorescing
molecules (as few as 50 molecules per cubic micron) can
be detected (6-8). In a given sample, through the use of
multiple staining, different probes will reveal the presence
of different target molecules. Although the fluorescence
microscope cannot provide spatial resolution below the
diffraction limit of the respective objectives, the presence
of fluorescing molecules below such limits is made
visible.
Techniques of fluorescence microscopy can be applied
to organic material, formerly living material, or to living
material (with the use of in vitro or in vivo fluorochromes).
These techniques can also be applied to inorganic material
(especially in the investigation of contaminants on
semiconductor wafers). There are also a burgeoning
number of studies using fluorescent probes to monitor
rapidly changing physiological ion concentrations
(calcium, magnesium, etc.) and pH values in living cells
(1, 7, 8).
There are specimens that fluoresce when irradiated
with shorter wavelength light (primary or
autofluorescence). Autofluorescence has been found
useful in plant studies, coal petrography, sedimentary rock
petrology, and in the semiconductor industry. In the study
of animal tissues or pathogens, autofluorescence is often
either extremely faint or of such non-specificity as to
make autofluorescence of minimal use. Of far greater
value for such specimens are the fluorochromes (also
called fluorophores) which are excited by irradiating light
and whose eventual yield of emitted light is of greater
intensity. Such fluorescence is called secondary
fluorescence.
Fluorochromes are stains, somewhat similar to the
better-known tissue stains, which attach themselves to
visible or sub-visible organic matter.
These
fluorochromes, capable of absorbing and then re-radiating
light, are often highly specific in their attachment
targeting and have significant yield in absorption-emission
ratios. This makes them extremely valuable in biological
Fluorescence Microscopy
Fluorescence microscopy is an excellent tool for
studying material which can be made to fluoresce, either
in its natural form (primary or autofluorescence) or when
treated with chemicals capable of fluorescing (secondary
fluorescence). This form of optical microscopy is rapidly
reaching maturity and is now one of the fastest growing
areas of investigation using the microscope (6-8).
Figure 26. Schematic diagram of the configuration of reflected
light fluorescence microscopy. Light emitted from a mercury
burner is concentrated by the collector lens before passing through
the aperture and field diaphragms. The exciter filter passes only
the desired excitation wavelengths, which are reflected down
through the objective to illuminate the specimen. Longer
wavelength fluorescence emitted by the specimen passes back
through the objective and dichroic mirror before finally being filtered
by the emission filter.
The basic task of the fluorescence microscope is to
permit excitation light to irradiate the specimen and then
to separate the much weaker re-radiating fluorescent light
from the brighter excitation light. Thus, only the emission
light reaches the eye or other detector. The resulting
fluorescing areas shine against a dark background with
sufficient contrast to permit detection. The darker the
background of the non-fluorescing material, the more
efficient the instrument. For example, ultraviolet (UV)
light of a specific wavelength or set of wavelengths is
produced by passing light from a UV-emitting source
through the exciter filter. The filtered UV light
illuminates the specimen, which emits fluorescent light
of longer wavelengths while illuminated with ultraviolet
light. Visible light emitted from the specimen is then
filtered through a barrier filter that does not allow reflected
UV light to pass. It should be noted that this is the only
35
OPTICAL MICROSCOPY
Davidson and Abramowitz
burdened with long exposures and a difficult process for
developing emulsion plates. The primary medium for
photomicrography was film until the past decade when
improvements in electronic camera and computer
technology made digital imaging cheaper and easier to
use than conventional photography. This section will
address photomicrography both on film and with
electronic analog and digital imaging systems.
The quality of a photomicrograph, either digital or
recorded on film, is dependent upon the quality of the
microscopy. Film is a stern judge of how good the
microscopy has been prior to capturing the image. It is
essential that the microscope be configured using Köhler
illumination, and that the field and condenser diaphragms
are adjusted correctly and the condenser height is
optimized. When properly adjusted, the microscope will
yield images that have even illumination over the entire
field of view and display the best compromise of contrast
and resolution.
application. The growth in the use of fluorescence
microscopes is closely linked to the development of
hundreds of fluorochromes with known intensity curves
of excitation and emission and well-understood biological
structure targets (6). When deciding which label to use
for fluorescence microscopy, it should be kept in mind
that the chosen fluorochrome should have a high
likelihood of absorbing the exciting light and should
remain attached to the target molecules. The
fluorochrome should also be capable of providing a
satisfactory yield of emitted fluorescence light.
Illustrated in Figure 27 is a photomicrograph of cat brain
cells infected with Cryptococcus. The image was made
using a combination of fluorescence and DIC microscopy,
taking advantage of the features from both contrast
enhancing techniques. Infected neurons are heavily
stained with the DNA-specific fluorescent dye acridine
orange, and the entire image is rendered in a pseudo threedimensional effect by DIC.
Photographic Films
Films for photography are coated with a light-sensitive
emulsion of silver salts and/or dyes. When light is allowed
to expose the emulsion, active centers combine to form
a latent image that must be developed by use of
photographic chemicals (19, 37-39). This requires
exposure of the film in a darkened container to a series
of solutions that must be controlled with respect to
temperature, development time, and with the appropriate
agitation or mixing of the solutions. The developing
process must then be halted by means of a stop solution.
Next, the unexposed emulsion material, which consists
of silver salts and dyes, is cleared and the film fixed, then
washed and dried for use. The development, stop, fixing,
and clearing must be done under darkroom conditions or
in light-tight developing tanks, and film must be handled
in complete darkness. The rigors of temperature, duration,
and agitation are usually dependent upon the film being
used. For example, the Kodachrome K14 process is far
more demanding than the E6 process used for Ektachrome
and Fujichrome (19, 37). The emulsion speed determines
how much light must be used to expose the film in a given
time period. Films are rated according to their ASA or
ISO number, which gives an indication of the relative film
speed. Larger ISO numbers indicate faster films with an
ISO of 25 being one of the slowest films available and
ISO 1600 one of the fastest. Because the microscope is
a relatively stable platform with good illumination
properties, films in the 50-200 ISO range are commonly
used for photomicrography.
Film is divided into a number of categories depending
upon whether it is intended for black/white or color
Figure 27. Fluorescence/DIC combination photomicrograph of
cat brain tissue infected with Cryptococcus. Infected neurons
are heavily stained with the DNA-specific fluorescent dye acridine
orange. Note the pseudo three-dimensional effect that occurs
when these two techniques are combined. The micrograph was
recorded using a 40x objective.
Photomicrography and Digital
Imaging
The use of photography to capture images in a
microscope dates back to the invention of the photographic
process (37). Early photomicrographs were remarkable
for their quality, but the techniques were laborious and
36
OPTICAL MICROSCOPY
Davidson and Abramowitz
available in color (ID numbers 668/669 or 58/59) or
black/white (ID numbers 667, 52, 665, 55, etc.).
The Polaroid large format films produce a paper
negative and a paper positive print. After the positive print
has been made, the paper negative is peeled away and
discarded. This can be accomplished within a matter of a
few minutes. The color films, with the exception of the
film pack 64T and the large format 64T, are balanced for
daylight (5500 Kelvin) color temperature. All
microscope manufacturers supply adapters for their
photomicrographic cameras that accept large format
Polaroid film sizes.
Polaroid black/white films 665 P/N and 55 P/N require
special mention. These films produce a paper positive
print and a polymer-based negative. If the negative is
bathed in an 18% sodium sulfite solution for a few
minutes, washed, and hung up to dry, it will produce a
permanent negative of high quality and high resolution
suitable for use in printing (37, 39).
The Polaroid 35mm films deserve more popular use.
They are loaded into the usual 35mm camera back and
exposed in the typical manner according to their rated
ISO and color temperature requirements. When the film
is purchased, the container includes the film cassette and
a small box of developer. The film is processed in a
tabletop processor that measures approximately 4" x 5" x
9" in size. All processing is carried out in daylight after
the processor has been loaded and closed. The finished
strip of positive color or positive black/white
transparencies is removed and ready for examination and
for cutting apart for mounting in frame holders for
projection. The actual processing takes only five minutes
and produces micrographs of quite accurate color and
good resolution.
Recently, Polaroid has marketed a relatively
inexpensive photomicrographic camera call the
MicroCam. This camera is lightweight and can be inserted
into one of the eyetubes or phototube of the microscope.
It contains an exposure meter, an eyepiece and an
automatically controlled shutter. The camera accepts 339
color film packs or 331 black/white film packs. These
films, approximately 3" x 3" in size, are self-developing
and do not require peeling apart. Although the films have
only about half the resolution of the more common large
format Polaroid films, the resulting print is easily
obtained and may be quite adequate for many applications.
photography. Color films are subdivided into two types:
color films that yield positive transparencies (colors like
those of the image being observed) and color negatives
(colors complementary to those in the microscope image,
e.g., green for magenta). The color films can be further
subdivided into two groups: films designed to receive light
of daylight quality and those designed to receive indoor
or tungsten light. As a rule, but with some exceptions,
transparency or positive films (slides) have the suffix
chrome, such as Kodachrome, Ektachrome, Fujichrome,
Agfachrome, etc. Color negative films usually end with
the suffix color, such as Kodacolor, Ektacolor, Fujicolor,
Agfacolor, etc. (37). Each of these film types is offered
in a variety of film speeds or ISO ratings. Film packages
usually display the ISO of the film and whether the film is
intended for daylight or an indoor/tungsten balanced
illumination. Modern film magazines have a code (termed
the DX number) that allows camera backs to automatically
recognize the film speed.
The color temperature of tungsten-halide lamps found
in modern microscopes varies between 2900K and
3200K, depending upon the voltage applied to the lamp
filament. Film manufacturers offer film balanced for this
illumination, and usually indicate on the magazine that the
film is intended for indoor or scientific use. Fuji offers
a very nice transparency film, Fujichrome 64T, that has a
rather slow emulsion speed intended for tungsten
illumination, but is designed to perform well with push
film processing. To push a film, it is first underexposed
by one or several f-stops, then the development time in
the first developer is increased to decrease film density.
This technique often will increase the color saturation of
the image (19, 37).
Daylight-balanced film, by far the most common film
available at retailers at a wide variety of ISOs, can also be
used with the microscope, provided an appropriate filter
is added to the light path. Manufacturers usually add a
daylight conversion light filter to their microscope
packages as standard equipment and high-end research
microscopes usually have this filter (called a daylight
color temperature conversion filter) in a cassette housed
in the base of the microscope. Almost any color print or
transparency film can be used for microscopy, provided
the daylight-balanced filter is in place for those films
designed for a 5500K color temperature or removed if
the film is balanced for tungsten illumination (3200K).
For many applications, securing the photomicrograph
almost immediately is a necessity or a great advantage.
Here the Polaroid films are unrivaled. These films for
photomicrography are available in three sizes: 35
millimeter Polachrome (color transparency), and Polapan
(black/white) in 3¼” x 4¼” film packs and 4” x 5”
individual film packets (37, 39). Larger formats are
Digital Photomicrography
Over the past decade, the quality of digital cameras
has greatly improved and today, the market has exploded
with at least 40 manufacturers offering a wide variety of
models to suit every application. The cameras operate by
37
OPTICAL MICROSCOPY
capturing the image projected directly onto a computer
chip without the use of film. In recent years, the number
of pixels of information capable of being captured and
stored by the best digital cameras is approaching, but still
short of, the resolution available with traditional film (37).
Digital images offer many opportunities for computercontrolled image manipulation and enhancement as well
as the advantage of permanence of digital storage. The
highest quality digital cameras can cost many thousands
of dollars.
Selection of an electronic camera must be preceded
by careful consideration of its proposed use. This
includes examination of fixed specimens or live
specimens, need for grayscale or true color images,
sensitivity to low light levels as in fluorescence,
resolution, speed of image acquisition, use in qualitative
or quantitative investigations, and the video feed rate into
a computer or VCR (1, 2, 9, 37).
The two general types of electronic cameras available
for microscopy are the tube cameras (Vidicon family) or
CCD (charge coupled device) cameras. Either type can
be intensified for increased sensitivity to low light. The
SIT (silicon intensified tube) or ISIT is useful for further
intensification of tube cameras, while the ICCD is an
intensified CCD camera. CCD cameras can also be cooled
to increase sensitivity by giving a better signal to noise
ratio. These kinds of cameras can be designed to respond
to light levels undetectable by the human eye (2, 37).
The criteria for selection of an electronic camera for
microscopy include sensitivity of the camera, quantum
efficiency, signal to noise ratio, spectral response,
dynamic range capability, speed of image acquisition and
readout, linearity or response, speed of response in
relation to changes in light intensity, geometric accuracy,
and ready adaptability to the microscope. A very important
criterion for the newer digital CCD cameras is resolution.
Current chips range from as few as 64 x 64 pixels up to
2048 x 2048 and above for very specialized applications,
but larger arrays are continuously being introduced and,
at some not-to-distant time, digital image resolution will
rival that of film.
The purchaser must also decide whether or not the
camera will operate at video rate (therefore being easily
compatible with such accessories as video recorders or
video printers). Tube cameras and CCD cameras are
available for video rate operation. CCD cameras can also
deliver slow acquisition or high-speed acquisition of
images. Scientific, rather than commercial grade, CCD
cameras are the variety most suitable for research.
CCD cameras are usually of small size. They have
low distortion, no lag, and good linearity of response.
These cameras are also more rugged and less susceptible
to mishandling than tube cameras. Each pixel of the CCD
Davidson and Abramowitz
camera serves as a well for charge storage of the incoming
photons for subsequent readout. The depth of these wells
relates to the quality of the response. Binning of adjacent
pixels by joining them together into super pixels can be
employed to speed readout in a slow-scan CCD camera
(2, 37).
An emerging technology that shows promise as the
possible future of digital imaging is the active pixel sensor
(APS) complementary metal oxide semiconductor
(CMOS) camera on a chip. Mass production of CMOS
devices is very economical and many facilities that are
currently engaged in fabrication of microprocessors,
memory, and support chips can be easily converted to
produce CMOS optical sensors. Although CCD chips
were responsible for the rapid development of video
camcorders, the technology has remained trapped as a
specialized process that requires custom tooling outside
the mainstream of integrated circuit fabrication. Also,
the CCD devices require a substantial amount of support
circuitry and it is not unusual to find five to six circuit
boards in a typical video camcorder.
In the center of the APS CMOS integrated circuit is a
large array of optical sensors that are individual
photodiode elements covered with dyed filters and
arranged in a periodic matrix. A photomicrograph of the
entire die of a CMOS chip is illustrated in Figure 28. High
magnification views of a single “pixel” element (Figure
28) reveal a group of four photodiodes containing filters
based on the primary colors red, green, and blue. Each
photodiode is masked by either a red, green, or blue filter
in low-end chips, but higher resolution APS CMOS
devices often use a teal (blue-green) filter in place of one
of the green filters. Together, the four elements
illustrated in Figure 28 comprise the light-sensitive
portion of the pixel. Two green filter masks are used
because visible light has an average wavelength of 550
nanometers, which lies in the green color region. Each
pixel element is controlled by a set of three transistors
and an amplifier that work together to collect and organize
distribution of optical information. The array is
interconnected much like memory addresses and data
busses on a DRAM chip so that the charge generated by
photons striking each individual pixel can be accessed
randomly to provide selective sampling of the CMOS
sensor.
The individual amplifiers associated with each pixel
help reduce noise and distortion levels, but they also
induce an artifact known as fixed pattern noise that arises
from small differences in the behavior of individual pixel
amplifiers. This is manifested by reproducible patterns
of speckle behavior in the image generated by CMOS
active pixel sensor devices. A great deal of research effort
has been invested in solving this problem, and the residual
38
OPTICAL MICROSCOPY
Davidson and Abramowitz
If you have prized transparencies and negatives
collected over a long period of time, many of the better
camera stores can take these and put them onto a Kodak
Photo CD, which is the same size as an audio CD. The
Photo CD can hold up to 100 images that are stored at
several levels of resolution. Digital images recorded onto
the Photo CD can be displayed on a good monitor by
means of a Photo CD player. If you have a computer and
a program such as Adobe Photoshop, Corel Photo Paint,
or Picture Publisher and a CD drive, you can open the
images on your computer screen, manipulate and/or
enhance the images and then print the images using a
digital printer—all without a darkroom! Kodak, Fuji,
Olympus, Tektronix, and Sony market dye sublimation
printers that can produce prints virtually indistinguishable
from those printed with the usual color enlarger in a
darkroom.
35mm negative and positive transparency scanners and
flatbed scanners, available from such manufacturers as
Nikon, Olympus, Polaroid, Kodak, Agfa, Microtek,
Hewlett-Packard, etc., can directly scan transparencies
or negatives or prints into your computer for storage or
manipulation. Images can be stored on the hard drive of
the computer or stored on floppy disks in JPEG or TIFF
files. Because floppy disks have storage limited to 1.44
megabytes, many micrographers are now storing images
on Zip disks or magneto-optical drive disks; these can
hold many images to sizes of 10 megabytes or more.
Another popular storage medium, quickly gaining
widespread popularity, is the recordable CD-ROM.
Magneto-optical disks or CD-ROMs can be given to
commercial printers and then printed with stunning color
accuracy and resolution.
A photomicrograph is also a photograph. As such, it
should not only reveal information about the specimen, it
should also do so with attention to the aesthetics of the
overall image. Always try to compose photomicrographs
with a sense of the balance of the color elements across
the image frame. Use diagonals for greater visual impact,
and scan the frame for unwanted debris or other artifacts.
Select a magnification that will readily reveal the details
sought. Remember to keep detailed records to avoid
repeating mistakes and to help with review of images that
are several years old. Excellent photomicrographs are
within the capability of most microscopists, so pay
attention to the details and the overall picture will
assemble itself.
level of noise has been dramatically reduced in CMOS
sensors. Another feature of CMOS active pixel sensors
is the lack of column streaking due to pixel bloom when
shift registers overflow. This problem is serious with the
CCDs found in most video camcorders. Another
phenomenon, known as smear, which is caused by charge
transfer in CCD chips under illumination, is also absent
in CMOS active pixel sensor devices.
Assisting the CMOS device is a coprocessor that is
matched to the APS CMOS to optimize the handling of
image data. Incorporated into the co-processor is a
proprietary digital video processor engine that is capable
of performing automatic exposure and gain control, white
balance, color matrixing, gamma correction, and aperture
control. The CMOS sensor and co-processor perform
the key functions of image capture, digital video image
processing, video compression, and interfacing to the
main computer microprocessor.
Figure 28. CMOS active pixel sensor used in new camera on a
chip technology that is rapidly emerging. The photomicrograph
captures the entire integrated circuit surface showing the photodiode
array and support circuitry. The insets illustrate progressively higher
magnification of a set of pixel elements and a single pixel element
composed of four dyed photodiodes.
The primary concerns with CMOS technology are the
rather low quality, noisy images that are obtained with
respect to similar CCD devices. These are due primarily
to process variations that produce a slightly different
response in each pixel, which appears in the image as snow.
Another problem is that the total amount of chip area that
is sensitive to light is less in CMOS devices, thus making
them less sensitive to light. These problems will be
overcome as process technology advances and it is very
possible that CMOS devices will eclipse the CCD as the
technology of choice in the very near future.
39
OPTICAL MICROSCOPY
Davidson and Abramowitz
21. H. W. Zieler, The Optical Performance of the Light Microscope
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26. R. McLaughlin, Special Methods in Light Microscopy.
Microscope Publications, Ltd., Chicago, 1977, 337 pp.
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Acknowledgements
The authors would like to thank the Molecular
Expressions (http://microscopy.fsu.edu) web design and
graphics team for the illustrations in the paper. We would
also like to thank George Steares, of Olympus America,
Inc., for providing advice and microscopy equipment used
in illustration of this manuscript. Assistance from Chris
Brandmaier, of Nikon USA, Inc., in providing equipment
for reflected light differential interference contrast
microscopy is also appreciated. Funding for this research
was provided, in part, by the National High Magnetic Field
Laboratory (NHMFL), the State of Florida, and the
National Science Foundation through cooperative grant
No. DMR9016241. We would also like to thank James
Ferner, Samuel Spiegel, and Jack Crow for support of the
Optical Microscopy program at the NHMFL.
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Optical Microscopy
Davidson and Abramowitz
41
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