Microscope Objectives - Wiley Online Library

Microscope Objectives - Wiley Online Library
Microscope Objectives
The basic task of a microscope is to provide
enlarged images of small objects. Of all the
optical components of the microscope, the objective is the most crucial to image formation.
The microscope objective must provide:
1. Magnification: an image that is enlarged
with respect to the specimen
2 Resolution: an image whose details are
clearly separated
3. Contrast or visibility: an image with sufficient contrast that its details are readily distinguishable from each other and from background material in the field of view when
viewed by the human eye or by a camera
4. Fidelity: an image that is a faithful reproduction of the original specimen, free from
distortion (aberration) and spurious detail (artifact).
IMAGE FIDELITY
Lens Aberrations
The image of the specimen projected by the
objective is an “optical replica” of the original,
suspended in space along the optical axis ~10
mm below the top of the eyepiece tubes (Figs.
2.2.1 and 2.2.2). The objective is constructed
to be as free as possible from aberrations that
would degrade the magnified image representing the original specimen. Aside from providing magnification, the objective must pro-
UNIT 2.2
duce an image that is, at each and every point
within it, a faithful mapping of the specimen in
all the following respects:
1. Specimen point to image point
2. Specimen line to image line
3. Specimen shape to image shape
4. Specimen flatness to image flatness
5. Specimen color to image color.
From the standpoint of geometric optics,
simple glass lenses may exhibit two main types
of aberrations: (1) aberrations related to wavelength or color, termed chromatic aberrations,
that occur when white (polychromatic) light is
used; and (2) aberrations that occur even when
monochromatic light (light of a single color or
wavelength) is used. It is important to understand that no objective, no matter how well-corrected for aberrations, can ever render an image
“point”—i.e., infinitesimal in size, like a true
point. Because of the wave nature of light and
the fact that light is scattered (diffracted) as it
traverses the minute pores and edges of a specimen, the best that even a perfectly corrected
objective can do is to represent a specimen point
as a tiny disk of light known as an Airy disk
(Fig. 2.2.3).
Light Refraction and Refractive Index
In the light microscope, light passes not only
through the specimen but also through a series
of glass lenses and accessories, and through air
eyepiece
10 mm
plane of intermediate
image at eyepiece
diaphragm
objective
specimen
Figure 2.2.1 Schematic diagram of a compound microscope, illustrating the site of intermediate
image formation at the plane of the eyepiece diaphragm. Reproduced from Abramowitz (1994) by
courtesy of Olympus America.
Image Cytometry
Instrumentation
Contributed by Mortimer Abramowitz and Marc M. Friedman
Current Protocols in Cytometry (1997) 2.2.1-2.2.14
Copyright © 1997 by John Wiley & Sons, Inc.
2.2.1
or another medium such as oil. As light passes
from one medium to another it is bent, or
refracted, to a degree that depends on the refractive indices of the adjacent media. The
refractive index, n, for a particular medium is
defined as the ratio of the speed of light in a
vacuum to the speed of light in that medium.
The slower the speed of light in a medium, the
greater its refractive index. For all practical
purposes the refractive index of air is 1—that
is, light travels at the same speed in air as in a
vacuum. All other media have refractive indices
>1. The refractive index of glass is ~1.5 and that
of water is ~1.33. When light passes from a less
dense medium (e.g., air; lower index of refraction) to a more dense medium (e.g., glass;
higher index of refraction) at an angle other
than 90o, the rays are bent (refracted) toward
the perpendicular. Conversely, light rays are
bent away from the perpendicular when travelling from a medium of higher refractive index
to a medium of lower refractive index. These
basic properties of light are central to the prac-
image
tical use of microscopes and to the design of
microscope lenses, which typically must gather
light passing through an aqueous specimen
(e.g., a cell) and thence through a glass cover
slip to air or another medium such as oil. For
example, the refractive index of standard microscope immersion oil is carefully set at 1.515
so as to match that of the glass in the cover slip
and the objective lens, to eliminate unwanted
refraction. More detailed discussion of these
properties may be found in the references listed
at the end of this unit.
Chromatic Aberrations
Chromatic aberrations result from the fact
that glass used in microscope lenses exhibits
different refractive indices at different wavelengths of light. Light of shorter wavelengths,
at the blue end of the spectrum, is brought to a
focus nearer to the back of the objective than
light of longer wavelengths (Fig. 2.2.4). This
effect is known as longitudinal chromatic aberration. Even when light of different wave-
Figure 2.2.2 Formation of a real image of a point by
an ideal lens. Reproduced from Abramowitz (1994) by
courtesy of Olympus America.
specimen
Figure 2.2.3 Airy disk diffraction image of a point,
illustrating the formation of the image of a specimen
point as a disk of light rather than a dimensionless
fine point. Reproduced from Abramowitz (1994) by
courtesy of Olympus America.
Microscope
Objectives
2.2.2
Current Protocols in Cytometry
lengths is brought to a common focus by employing optical correction, the image of a point
in the outer part of the field of view may be
magnified to a greater extent with blue light
than with red. This is known as chromatic
difference of magnification, or lateral chromatic aberration, and causes the image of a
point in white light to appear ringed by colors.
These errors are typically corrected by employing glasses with differing optical properties,
such as flint glass, crown glass, and low-dispersion glasses, in various lens elements within the
objective. Manufacturers may use different design methods to correct for these aberrations.
Older-style microscopes used compensating
eyepieces to correct for chromatic difference of
magnification. Olympus and Nikon now correct for chromatic difference of magnification
within the objective itself, whereas Leica and
Zeiss accomplish this correction by means of a
tube lens built into the microscope stand.
Aberrations in Monochromatic Light
The most serious aberration in monochromatic light is spherical aberration. Light passing through the outer region of a convex lens is
brought to a focus nearer to the back of the
objective than light passing through the center
of the lens (Fig. 2.2.5). The net result is a
blurring of the image, because a single point in
the specimen is represented by a series of points
in the image, with each image point focused at
a slightly different distance from the back of
the objective. These and other monochromatic
aberrations are corrected by designing required
lens elements of different shapes and forming
these into compound elements by cementing
them into doublets and triplets. In practice,
red focus
green focus
violet-blue
focus
Figure 2.2.4 Chromatic
aberration in an uncorrected lens,
illustrating the existence of
different focal planes for different
wavelengths of light. Reproduced
from Abramowitz (1994) by
courtesy of Olympus America.
objective
white light
axial rays
peripheral rays
uncorrected lens
monochromatic light
Figure 2.2.5 Spherical
aberration, or the difference of
focus for light rays traversing the
outer zones of a lens compared
to light passing more centrally.
Reproduced from Abramowitz
(1994) by courtesy of Olympus
America.
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Instrumentation
2.2.3
Current Protocols in Cytometry
eliminating spherical aberration is particularly
difficult. Despite the excellent design of modern objectives, a microscopist may inadvertently introduce spherical aberration by using
a too-thick layer of mounting medium below
the cover slip and/or a cover glass of incorrect
thickness (see discussion of Special Features
later in this unit) or by employing improperly
corrected optical accessories in the light path.
Accessory image-forming lenses such as eyepieces, relay lenses, and tube lenses are used to
enlarge, correct, or focus an image projected by
the objective lens, but they must be carefully
designed and properly used so as not to degrade
the image. Spherical aberration is particularly
troublesome when taking a series of optical
sections through living cells or tissues for threedimensional reconstruction, in which dozens or
hundreds of images may be recorded by successively focusing to great depths within a
tissue, using intervals as small as 0.5 µm. Internal organelles encountered in the focal plane
of each section may introduce, unpredictably,
regions of differing indices of refraction that
cannot be anticipated in the design of the lens.
Other aberrations observed using monochromatic light include coma, astigmatism, distortion, and curvature of field. These errors
must be corrected in the design of the glass
elements of the objective and are not under the
control of the user. With coma, the image of a
point appears comet-shaped (Fig. 2.2.6). With
astigmatism, the image of a point may appear
as a line with either a vertical or horizontal
orientation (Fig. 2.2.7). Distortion (Fig. 2.2.8)
causes images of parallel lines to bow outward
(barrel distortion) or inward (pincushion distortion). With curvature of field (Fig. 2.2.9), an
error inherent to curved lens elements, the image may be in focus in the center of the field of
view but not at the edges, or vice versa. Modern
objectives, even those from the less expensive
series of the major manufacturers, are corrected
to compensate for these aberrations.
PROPERTIES OF MICROSCOPE
OBJECTIVES
Numerical Aperture
Numerical aperture (NA) is a term devised
by Ernst Abbe in the late 19th century to describe the light-gathering ability of an objective. Figure 2.2.10 represents a set of longitudinal slices through the cone of light emanating
from a point in the specimen and captured by
each of three objective lenses. If the included
image plane
axis
objective
specimen point
Microscope
Objectives
Figure 2.2.6 Diagrammatic representation showing the comet-like smearing of an image point
from a lens that has not been corrected for coma aberration. Reproduced from Abramowitz (1994)
by courtesy of Olympus America.
2.2.4
Current Protocols in Cytometry
half angle is designated µ, then the NA is
defined as n × sin µ, where n is the refractive
index of the medium between the cover slip and
the objective. For dry objectives, with air between the cover slip and the objective, the
maximum achievable NA is 0.95. Modern oilimmersion objectives offer a maximum NA of
1.4, whereas water-immersion objectives offer
a maximum NA of 1.25.
Resolving Power
The importance of achieving the highest
possible NA becomes evident upon investigating the theoretical resolving power of a lens,
which may be described as its ability to reveal
a separation between two closely spaced objects. The theoretical resolving power is the
maximum separation that is achievable, and
depends on two factors: the wavelength of the
Ib
I
image plane
Ia
principal axis
objective
specimen point
Figure 2.2.7 Astigmatism, an aberration in the outer lens zones caused by unequal magnification
in the different azimuths. A specimen point appears in the image not as a point but as a line.
Reproduced from Abramowitz (1994) by courtesy of Olympus America.
specimen
image with
barrel distortion
image with
pincushion
distortion
Figure 2.2.8 Barrel and pincushion distortion of parallel lines in an uncorrected lens. Reproduced
from Abramowitz (1994) by courtesy of Olympus America.
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Current Protocols in Cytometry
illuminating light and the numerical aperture
of the optical system (objective and condenser).
The formula devised by Abbe for determining
resolving power in transmitted light microscopy is:
λ illuminating light
D=
NA objective + NA condenser
where D is the distance between two close-lying objects that are nevertheless separable by
eye. The highest theoretical resolving power of
the transmitted light microscope, using an objective and condenser each having an NA of
1.4, can reveal a separation of 0.2 µm using
green light.
CONSTRUCTION AND TYPES OF
MICROSCOPE OBJECTIVES
The construction of microscope objectives
must ensure that optical alignment of the glass
elements within the casing of the objective is
curved image at
image plane
objective
flat specimen
Figure 2.2.9 Curvature of field, in which the image of a flat specimen is curved and therefore not
in a uniform plane of focus. Reproduced from Abramowitz (1994) by courtesy of Olympus America.
µ
oil
low-NA
dry
objective
NA = n sin µ
Microscope
Objectives
higher-NA
dry
objective
oil
highest-NA
oil-immersion
objective
Figure 2.2.10 Illustration of the light gathering ability of three lenses with increasing numerical
aperture (NA), where n is the refractive index of the medium between the objective and the cover
slip and µ is the half-angle of the cone of light captured by the objective. Reproduced from
Abramowitz (1994) by courtesy of Olympus America.
2.2.6
Current Protocols in Cytometry
highly accurate, and must provide maximum
protection for exposed glass surfaces. Accurate
mounting and spacing of the elements is critical
to proper optical performance, and is typically
accomplished using tiny metal spacer rings.
The exposed front surface of the objective lens
and the internal glass surfaces in contact with
air spaces between lens elements are coated
with special thin deposits that greatly reduce
internal glare to maintain a high throughput of
light. Objectives with very short working distances—e.g., those that must be brought very
close to the specimen in order to focus properly—are often designed with a spring-loaded
tip that retracts the front lens into the barrel if
the objective is inadvertently brought into contact with the top of the cover glass.
Objectives are classified and labeled according to their degree of optical correction. The
most common, in order of increasing correction
and cost, include achromats (marked Ach, or
sometimes unmarked), semiapochromats or
fluorites (Fl), and apochromats (Apo). An objective may also be inscribed with the designation Plan (e.g., Plan Apo) indicating correction
for flatness of field, which ensures that the
image remains in focus from one edge of the
field of view to the other. Plan objectives are
especially important when the entire field of view
must be critically examined without refocus-
ing, and when recording images using photomicrography or video archiving.
High-power objectives that have high NA
and offer excellent correction (e.g., Plan Apos)
may contain more than ten glass components,
many of which are doublets or triplets (Fig.
2.2.11). The finished objective must possess
both mechanical stability and heat stability
within the normal temperature range encountered in the laboratory, and must tolerate normal
handling. However, the large number of closely
spaced and precisely positioned elements creates a practical problem: such objectives will
not suffer the excesses tolerated by less complex objectives. Dropping or severely bumping
a Plan Apo objective, or leaving it in a car trunk
on a hot summer day, will likely result in cracking and/or loosening of the glass elements and
may necessitate complete reassembly at the
factory, or replacement.
The metal jacket of an objective has inscriptions that provide the user with important information about it (Fig. 2.2.12). Written in a
simple, almost universal code, the markings
convey the magnification, tube length, type of
optical correction (finite or infinity-corrected),
numerical aperture, allowed cover glass thickness (a critical design criterion), focal length of
the tube lens (for infinity systems), and a classification of the objective according to the ex-
Figure 2.2.11 Schematic, longitudinal
section through a 60× Plan Apo objective
lens, illustrating the numerous and
complex elements of a highly corrected
objective. Diagram reprinted courtesy of
Olympus America.
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OLYMPUS
plan apochromat
numerical aperature
magnification
UPlanApo
immersion fluid
40x /1.0 oil iris
/0.17
infinity correction
corrected for coverglass
thickness of 0.17 mm
manufacturer
color-coded band
1.0>
<0.5
special feature:
dark-field iris
Figure 2.2.12 Diagrammatic representation of typical markings found on the case of an objective
lens.
tent of correction. Also, it is now standard to
inscribe a colored ring toward the front of the
barrel that tells the user the magnification just
at a quick glance (e.g., yellow for 10×, green
for 20×, blue for 40×, and white for 100×). An
additional colored or black ring may indicate a
phase objective or an immersion objective.
Some inscriptions may also be in color to signify that the objective is intended for specialized use, such as phase-contrast or polarization
microscopy.
MODERN OBJECTIVES
Achromats, Fluorites, and
Apochromats
Microscope
Objectives
Achromats are corrected to bring at least two
colors of light, usually red and blue, to a common focus and are most highly corrected for
spherical aberration in the apple-green portion
of the spectrum. Thus, when a green filter is
used in the illumination path, modern achromats will yield quite good images. Recently
designed achromats may have chromatic correction for three wavelengths and spherical
correction for two. They are well suited for use
in the clinical laboratory and do a creditable job
of providing good color photomicrographs.
However, the more rigorous demands of research microscopy usually mandate the use of
fluorites or apochromats.
Fluorite objectives are corrected chromatically and spherically for at least two colors
(blue and green) and more recently for a third
(red). Plan fluorites yield very good images in
photomicrography using white light and typical laboratory stains, and make good allaround objectives for both routine and more
demanding applications because they are wellcorrected, offer high numerical apertures,
and—at about two to four times the cost of an
achromat—are still significantly less expensive
than Plan Apo objectives.
Apochromats offer the highest available degree of correction and consequently are the
most expensive objectives, typically about ten
times the cost of an achromat and three to four
times the cost of a fluorite. Because of its high
degree of optical correction, an apochromat
usually has a numerical aperture that is much
higher than that of an achromat of comparable
magnification but only somewhat higher than
that of a comparable fluorite. Modern Apos are
chromatically corrected for at least four wavelengths (violet, blue, green, and red) and spherically corrected for three or four wavelengths.
Modern Apos and fluorites are typically Plancorrected because they are often used for demanding applications and image recording via
film or video techniques.
For those who wish to compare the quality
of images produced by different types of objectives, manufacturers offer various test slides
including a metallized slide with tiny pinholes,
a slide with fine lines spaced close together, and
a slide containing mounted diatoms whose different species have structural markings of a
specific fineness or periodicity. In former years,
the so-called “Abbe test plate” was used to test
for chromatic as well as spherical aberration,
2.2.8
Current Protocols in Cytometry
and its use is described in standard microscope
texts. For many users, a familiar stained slide
with an appropriate specimen can serve as a
suitable routine test object.
expert user may determine through rigorous
testing that a specific objective from one manufacturer can be used on another manufacturer’s
instrument to deliver superior performance for
a specific application.
Optical Correction and Tube Length
Objectives may be further classified as finite
(or finite-tube-length) objectives or infinitycorrected objectives according to the way in
which they are designed to project images. The
barrels of finite-type objectives are marked with
the mechanical tube length, defined as the distance in millimeters along the optical path from
the opening of the nosepiece to the top of the
observation tube where the eyepiece is inserted.
This number is most commonly 160 (mm).
Infinity-corrected objectives are inscribed with
the symbol ∞ to signify such design; they may
also be inscribed with the focal length of the
tube lens employed in the system, which forms
the intermediate image (Figs. 2.2.13 and
2.2.14).
Finite objectives project an image that converges to a focus at the plane of the fixed
diaphragm of the viewing eyepiece (Figs. 2.2.1,
2.2.15). If an optical accessory, such as a polarizing intermediate piece or fluorescence illuminator, is interposed between the back of the
objective and the eyepiece, it must incorporate
correcting lenses to return the image to proper
focus at the prescribed position in the plane of
the eyepiece diaphragm. Infinity-corrected objectives are designed to project the image of the
specimen “to infinity,” rather than to a fixed
plane within the eyepiece, and so light rays
arriving from all azimuths emerge from the
objective in parallel bundles (Fig. 2.2.14). To
bring the image to focus at the plane of the
eyepiece diaphragm, the microscope must incorporate a tube lens in the light path. Some
manufacturers mount the tube lens within the
body of the microscope, whereas others build
the tube lens into the binocular or trinocular
observation-tube head. With infinity correction, accessories interposed between the objective and the tube lens are far simpler to design
and are far less prone to introduce aberrations.
It is important to recognize that users should
not employ finite objectives on an infinity-designed stand, nor use infinity-corrected objectives on finite-designed stands. Furthermore, it
is not advisable to use infinity-corrected objectives interchangeably even among infinity-designed microscopes from different manufacturers, as the focal lengths of the tube lenses
differ and the various chromatic corrections are
achieved in different ways. Despite this, an
Immersion Objectives
Most biological objectives, as opposed to
reflected-light metallurgical objectives, are designed to properly correct for aberrations only
when used with a cover-slipped specimen, as
indicated by specific markings on the lens barrel (Fig. 2.2.12). Unless otherwise labeled, objectives are designed with the assumption that
air will occupy the space between the front lens
of the objective and the cover glass of the
specimen. Such objectives are called dry objectives, and have a maximum achievable NA of
0.95. In order to increase the numerical aperture
to a range of 1.0 to 1.4, an intermediate or
immersion fluid with a specific refractive index
must uniformly fill the space between the top
of the cover glass and the front lens of the
objective, and be in contact with both. Because
resolving power is directly proportional to NA,
achieving the highest resolution requires an
immersion-type objective, and the usual immersion medium is an oil with a refractive index
of 1.515, which is close to that of glass. Other
immersion objectives for specific applications
are designed for use with water or, less commonly, glycerin (glycerol) as the immersion
medium. Water-immersion objectives designed
for use with and without a cover slip have
become increasingly important in fluorescence
and confocal microscopy for the study of living
cells, and for three-dimensional reconstruction
of cells and tissues. These objectives help to
avoid the introduction of severe spherical aberration that may be incurred when using different
immersion media above and below the cover
slip, as when observing cells and tissues (in
water) with oil-immersion objectives. Spherical
aberration becomes more pronounced as the
user focuses deeper into the tissue and farther
below the cover slip. Immersion objectives are
always inscribed with the name of the required
immersion medium, and will yield a very distorted image if used dry or with the wrong
immersion fluid. Some objectives are designed
and marked for use with multiple immersion
media, such as water, oil, and glycerin, with a
correction collar that the user must be sure to
adjust to the proper setting for each medium.
Air bubbles must be scrupulously avoided
when using immersion fluids, as the contained
air has a significantly different refractive index
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Current Protocols in Cytometry
eyepiece diaphragm
plane
tube lens
objective
specimen
front focal plane
of objective
Figure 2.2.13 Diagrammatic representation of an infinity-corrected objective system. The diagram
illustrates parallel beams emerging from different azimuths (shaded) of an infinity-corrected
objective being brought to focus by a tube lens. Reproduced from Abramowitz (1994) by courtesy
of Olympus America.
than the immersion medium. It is also important
not to mix immersion fluids from different
manufacturers even if they have the same refractive index, as they will likely have quite
different viscosities and will therefore not mix
together adequately, but form optically refractive swirls that will interfere significantly with
proper image formation.
Special Features
Microscope
Objectives
In some applications—such as when viewing cells grown in thick-walled culture vessels—it may be physically impossible for the
objective to get close enough to the specimen
to focus properly. Manufacturers have therefore
designed dry “long-working-distance” (LWD)
objectives, most often used on inverted microscopes that can bring a specimen into focus
even when the distance to the specimen is ≥1
mm. Such objectives are usually inscribed as
LWD or ULWD (ultra-long-working-distance)
and may be specially designed to compensate
for a defined thickness of intervening glass or
plastic.
Although most biological objectives are designed for use with cover-slipped specimens,
some objectives are optically corrected for use
with non-cover-slipped specimens such as
blood smears. If so designed, the objective will
be inscribed with a “0” or “–” in place of a cover
glass thickness value (e.g., 0.17). Typical inscriptions would therefore be “160/0” or “160/–
” for the finite type or “∞/0” or “∞/–” for the
infinity type. If such an objective with a magnification >10× is used with a covered specimen, the image quality will be poor. At a magnification of ≤10×, objectives designed for use
with and without cover slips may be used interchangeably for routine applications.
Microscope cover glasses come in several
thicknesses, as indicated by a number (e.g., 1,
1.5, or 2). Each number represents a defined
2.2.10
Current Protocols in Cytometry
eyepiece
integrated
observation
tube
intermediate
image
tube lens
infinity
space
parallel
light
beam
intermediate
attachments
objective
Figure 2.2.14 Infinity space: the distance between the back of an infinity-corrected objective and
the tube lens (schematic). Reproduced from Abramowitz (1994) by courtesy of Olympus America.
thickness range. For example, #1.5 cover
glasses are typically 0.16 to 0.19 mm in thickness. Typical dry biological objectives are designed and optically corrected for a cover-glass
thickness of precisely 0.17 mm. Dry objectives
with NA greater than ~0.75 will suffer noticeable image degradation if the cover glass differs
even by a few hundredths of a millimeter from
the specified thickness. Because cover glass
thickness may vary by several hundredths of a
millimeter even within a package, “high dry”
(40× high-NA) objectives are available with an
adjustable correction collar and scale that permits them to be adjusted for different cover
glass thicknesses (e.g., from 0.11 to 0.23 mm).
Turning the correction collar to match the actual thickness of the individual cover glass in
use prevents the introduction of spherical aberration and its consequence, image degradation.
Using too much mounting medium on the tissue
will create an additional “cover glass–like”
optical layer that must be added to the thickness
of the cover glass to determine the total “effective” cover glass thickness. If the effective
cover glass thickness is different from that
specified for the objective, spherical aberration
will be introduced into the image. For this
reason, many experienced microscopists do not
rely on the correction collar’s numbered scale
to set the proper correction. Rather, they choose
a suitable area of the specimen and repeatedly
refocus the microscope while moving the correction collar to different positions, finally reaching the setting that provides the best image.
On objectives used for inverted tissue culture studies with flasks or other relatively thick
culture vessels, the correction collar may have
a range of correction from 0 to 2 mm; on
standard upright microscope objectives, the
range is usually from 0.11 to 0.22 mm.
OBJECTIVES FOR OTHER
MICROSCOPY APPLICATIONS
Phase Contrast
For phase-contrast microscopy, an annular
“phase plate” is installed by the manufacturer
inside the back of the objective. This plate
serves to “speed up” the undiffracted light passing through it and also to reduce its intensity.
Phase specimens, such as unstained cells and
tissues, are almost invisible in standard brightfield microscopy. The phase plate in the objective, when aligned with the annular opening of
a phase condenser, optically renders small
phase objects visible without the use of stains.
Because phase-contrast observation is often
done through glass or plastic culture vessels,
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2.2.11
Current Protocols in Cytometry
eyepiece diaphragm
plane
objective
front focal plane
of objective
specimen
Figure 2.2.15 Objective system of finite tube length, showing the projection of the image by a
finite objective to the intermediate image plane within the eyepiece tube. Reproduced from
Abramowitz (1994) by courtesy of Olympus America.
some manufacturers offer interchangeable accessory lenses or “caps” that attach to the front
lens of the objective (one set for use with plastic
vessels, one set for glass vessels) to avoid distortion of images.
Polarization Techniques
Microscope
Objectives
In polarization microscopy, it is important
that the objective itself not contribute to the
alteration of polarization effects induced by
the specimen. Because glass that is physically
strained affects polarized light, microscope
manufacturers carefully select objectives in
which the glass elements and their mountings
are strain-free. The barrel of strain-free objectives supplied with polarizing microscopes is
usually marked with a “P”, “SF,” or “POL” and
is sometimes inscribed in a color different from
the usual inscription color.
Differential interference contrast (DIC) microscopy is also invaluable for making small
phase objects readily visible. It has further
advantages in that it (1) yields a pseudo-three-
dimensional image, in which the object appears
shadowed—brighter on one side and darker on
the other—displaying “elevations” and “depressions” within the specimen; (2) permits the
use of high-NA optics; and (3) makes possible
“optical staining” and “optical sectioning” of
the specimen. In DIC microscopy, the distance
from the back focal plane of the objective to the
upper Wollaston prism (a special prism positioned above the objective) is usually critical,
and microscope companies may therefore designate particular objectives for use in DIC microscopy. These objectives are relatively strainfree, because interference microscopy also involves the use of polarized light, and may be
labeled DIC or NIC (for Nomarski interference
contrast, a particular type of DIC).
Dark-Field Microscopy
In transmitted-light dark-field microscopy,
the illumination is directed obliquely so that the
specimen appears bright on a dark background.
For dark-field microscopy with high-NA objec-
2.2.12
Current Protocols in Cytometry
tives (≥1.00), the NA of the objective must be
reduced below that of the oil darkfield condenser. Manufacturers therefore provide highNA objectives with built-in iris diaphragms
(see Fig. 2.2.12). For dark-field use, the diaphragm is closed down to yield an NA below
1.1. For general use, the diaphragm must be
fully open or optical performance will be degraded.
Ultraviolet (UV), Fluorescence, and
Infrared (IR) Applications
Standard glass objectives are relatively
opaque to wavelengths in the lower UV range,
below ∼380 nm. Special objectives are manufactured with special glasses to achieve greater
transmission of these lower wavelengths,
which are used to excite certain fluorescent
dyes for measurement of intracellular ions. The
cements used in complex lens elements for
fluorescence microscopy are nonfluorescing,
and the best fluorescence objectives are made
using quartz optics.
Other investigations may be carried out using longer, IR wavelengths (>750 nm), which
offer poorer resolution (see Abbe’s equation in
the discussion of Resolving Power) but greater
depth of penetration into biological (and other)
materials. Several companies offer objectives
specially designed to more efficiently transmit
wavelengths up to 1800 nm. The technical
departments of the major microscope companies can provide transmission and spectral data
for their objectives upon request to aid in selecting the proper objectives for special applications.
OTHER CONSIDERATIONS IN
CHOOSING OBJECTIVES
Other considerations may prove valuable in
understanding the performance of objectives
and in guiding the selection, purchase, and use
of suitable objectives.
Numerical aperture, the ability of the objective to capture a cone of light of wider angle,
has a crucial effect on resolution. Although
intuitively it may seem that resolving power
should increase with increasing magnification,
it can be shown that the ability to distinguish
closely spaced details within a specimen is
directly proportional to the twice the working
NA. However, the use of objectives with higherthan-necessary magnification and NA for a
given application can be detrimental not only
because they are more expensive, but also because the specimen area observed within a field
of view will be smaller and both the depth of
field (the vertical distance above and below the
plane being observed that is still in acceptable
focus) and working distance are shallower.
When the finest specimen details need to be
observed, high-NA objectives are required.
High-NA objectives are also indicated when
maximum throughput of light is needed. The
light transmittance for an objective, using visible wavelengths, typically varies with the
square of the NA of the objective. In reflectedlight and epifluorescence microscopy, light
passes through the objective twice (first the
illuminating light, and then the reflected or
fluorescent signal), and so the intensity varies
with the fourth power of the NA. In situations
where the light level is low, NA is a critical
factor in obtaining brighter images.
A question often asked is why higher magnification cannot be achieved simply by using
higher-magnification eyepieces with a given
objective. Because of limitations due to the size
of light waves themselves and the phenomenon
of diffraction, higher and higher magnifications
unaccompanied by increased NA will result in
images that are less and less clear. The limiting
factor in ensuring usable, as opposed to empty,
magnification is the NA of the objective (more
precisely, the average NA of the objective and
the condenser). Eyepieces and accessory lenses
are designed for use with certain objectives and
condensers, and should not be switched to increase magnification except as recommended
by the manufacturer. An oft-cited rule of thumb
is that the user should limit the total optical
magnification (the objective magnification
multiplied by the eyepiece magnification and
that of any other lenses) to between 500 and
1000 times the NA of the objective. At <500
times the NA, fine specimen details may not be
perceivable by the eye; at >1000 times the NA,
the likely result is empty magnification. In the
favored method of Koehler illumination, the
condenser diaphragm is partially closed down,
slightly lowering the overall NA in order to
improve contrast. Hence, a total magnification
of ∼750 times the NA will usually produce
excellent images with satisfactory contrast.
All of the foregoing discussion of objective
design, features, and performance assumes that
the optics (and the rest of the microscope)
remain forever in the pristine state in which they
presumably arrived. Proper care of the objectives, including handling, storage, and cleaning, are essential prerequisites to keeping them
in proper working order. The authors have often
noted that the best microscopy is not necessarily performed by those with the best equipment,
Image Cytometry
Instrumentation
2.2.13
Current Protocols in Cytometry
and quite often the performance of superior
optics is profoundly or subtly degraded by a
lack of care in choosing and maintaining the
optics.
Delly, J.G. 1988. Photography Through The Microscope. Eastman Kodak, Rochester, N.Y.
Inoue, S. 1986. Video Microscopy. Plenum Press,
New York.
LITERATURE CITED
Leitz, E. 1938. The Microscope And Its Application.
Ernst Leitz, Wetzlar, Germany.
Abramowitz, M. 1994. Optics: A Primer. Olympus
America Inc., New York.
Mollring, F.K. 1976. Microscopy From The Very
Beginning. Carl Zeiss, Oberkochen, Germany.
KEY REFERENCES
Spencer, M. 1982. Fundamentals of Light Microscopy. Cambridge University Press, Cambridge,
UK.
Abramowitz, M. 1985. Microscope Basics and Beyond. Olympus Corporation, New York.
Abramowitz, M. 1987. Contrast Methods in Microscopy: Transmitted Light. Olympus Corporation,
New York.
Abramowitz, M. 1993. Fluorescence Microscopy:
The Essentials. Olympus America Inc., New York.
Abramowitz, 1994. See above.
Bradbury, S. 1984. An Introduction to the Optical
Microscope. Oxford University Press, Oxford,
UK.
Contributed by Mortimer Abramowitz
Olympus America Inc.
Melville, New York
Marc M. Friedman
AccuMed International
Chicago, Illinois
Microscope
Objectives
2.2.14
Current Protocols in Cytometry
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