Introduction to the Microscope
1
Basic Microscope Parts
Introduction to the Microscope
3
Basic Microscope Parts
To the neophyte, using a microscope might
seem simple. All one has to do is turn on the
illuminator, place a specimen on the stage,
center it, focus it, and view. Unfortunately,
it’s not always that simple. Many microscope
adjustments require finesse and experience.
Nonetheless, a new user can easily learn
the proper illumination techniques and correct operation of the microscope to get good
results. This book will present both basic and
advanced techniques for using a microscope,
along with information about different types
of specimens, their preparation, and the best
way to view them. As with most things, simple
techniques yield the most basic images, while
more advanced techniques reveal hidden wonders in the microscopic world.
This chapter introduces the microscope by
describing the controls used most frequently.
Chapter 2 covers the parts of the microscope
in greater practical and theoretical detail.
Taken together, these chapters provide the
basics to get immediate results. When later
chapters cover more sophisticated steps and
techniques, readers will be able to build on
their basic knowledge and understanding to
perform the new techniques successfully.
Eyepiece
Coarse
Focusing
Knob
Body
Fine
Focusing
Knob
Stage
Condenser
Condenser
Focusing
Knob
Figure 1-1: A monocular microscope made by
Reichert in the 1930s. This is a typical instrument
from the early 1900s. Focusing is accomplished by
raising or lowering the microscope body that holds
the objectives and eyepiece. When a camera is
mounted over the eyepiece, its weight can stress the
focus controls. Typically, to prevent a downward drift
in focus, a friction clutch is tightened on the coarse
The cerebellum is used to control equilibrium in man.
focus. Turning this knob becomes more difficult,
This photograph taken with a 4x objective and 1x eye-
and for this reason, a microscope of this type is
piece shows the general structure of this tissue. It is
unsuitable for digital photomicrography.
stained with a dye combination that colors nuclei a
deep purple and the cytoplasm of the cell pink. By
using Köhler illumination, it is possible to light the
specimen so that the resultant micrograph has high
contrast and the field is evenly illuminated.
1
Basic Microscope Parts
Introduction to the Microscope
3
Basic Microscope Parts
To the neophyte, using a microscope might
seem simple. All one has to do is turn on the
illuminator, place a specimen on the stage,
center it, focus it, and view. Unfortunately,
it’s not always that simple. Many microscope
adjustments require finesse and experience.
Nonetheless, a new user can easily learn
the proper illumination techniques and correct operation of the microscope to get good
results. This book will present both basic and
advanced techniques for using a microscope,
along with information about different types
of specimens, their preparation, and the best
way to view them. As with most things, simple
techniques yield the most basic images, while
more advanced techniques reveal hidden wonders in the microscopic world.
This chapter introduces the microscope by
describing the controls used most frequently.
Chapter 2 covers the parts of the microscope
in greater practical and theoretical detail.
Taken together, these chapters provide the
basics to get immediate results. When later
chapters cover more sophisticated steps and
techniques, readers will be able to build on
their basic knowledge and understanding to
perform the new techniques successfully.
Eyepiece
Coarse
Focusing
Knob
Body
Fine
Focusing
Knob
Stage
Condenser
Condenser
Focusing
Knob
Figure 1-1: A monocular microscope made by
Reichert in the 1930s. This is a typical instrument
from the early 1900s. Focusing is accomplished by
raising or lowering the microscope body that holds
the objectives and eyepiece. When a camera is
mounted over the eyepiece, its weight can stress the
focus controls. Typically, to prevent a downward drift
in focus, a friction clutch is tightened on the coarse
The cerebellum is used to control equilibrium in man.
focus. Turning this knob becomes more difficult,
This photograph taken with a 4x objective and 1x eye-
and for this reason, a microscope of this type is
piece shows the general structure of this tissue. It is
unsuitable for digital photomicrography.
stained with a dye combination that colors nuclei a
deep purple and the cytoplasm of the cell pink. By
using Köhler illumination, it is possible to light the
specimen so that the resultant micrograph has high
contrast and the field is evenly illuminated.
4
Chapter 1
Basic Microscope Parts
Digital SLR
Camera Adapter Tube
Eyepiece
Trinocular Head
Nose piece
Magnification
Changer Optovar
Objective
Stage
Condenser
Condenser Focus
Field Diaphragm
Focus, Coarse
Focus, Fine
Figure 1-2: A Zeiss WL equipped with a digital SLR
camera. This microscope was manufactured in the
1960s and shows the design changes that made
the microscope more suitable for photography.
The microscope body has a trinocular head with
the vertical tube dedicated to holding the camera:
a microscopist can view a sample through the
eyepieces and conveniently record observations
with the camera. Note how the microscope body
is fixed in position: raising and lowering the stage
accomplishes focus.
There are three functional divisions of the
basic microscope controls: the first is holding the specimen, the second is focusing to
attain a sharp image, and the third is adjusting
the illumination to minimize glare (figures
1-1, 1-2, and 1-3). The stage supports the
specimen, which in turn is mounted on a glass
slide. While appearing simple, the stage is a
precisely engineered flat plate located in the
mid-region of the microscope. Its smooth
surface is perpendicular to the viewing optics,
and it is finely polished to allow minute movements of the slide without any friction or
sticking.
The slide is typically a glass rectangle, 1 mm
thick and 25 mm wide by 75 mm long. It is
made of an optical-grade glass with its surfaces parallel and polished. A sample, which
for many users is a thin slice of tissue that has
been stained with dyes, is placed on its upper
surface. A thin glass cover (coverglass) of a
defined thickness is placed over the sample,
sandwiching it between the slide and the coverglass. The resulting mounted slide is placed
on the stage with the coverglass facing up.
Two clips, one spring-loaded, grip the slide
securely. Two precise mechanical gears move
the clips vertically and horizontally on the
stage to position the specimen for viewing.
The microscopist focuses the specimen by
moving it closer or farther from the viewing
optics. In older microscopes, the slide is fixed
and the viewing optics are raised or lowered
(figure 1-1). On modern research microscopes,
the entire stage assembly is moved while the
optical train is fixed (figures 1-2 and 1-3).
Focusing by moving the stage assembly is
preferred when using a digital camera. Otherwise, the focusing mechanism has to carry
the weight of the camera as well as the microscope head. This additional weight can make it
difficult to turn the coarse focus control. For
the purposes of this book, I assume that the
reader is using a modern microscope with a
focusing stage.
There are two focusing controls: coarse
and fine. Typically, the coarse focus knob is
larger in diameter and is found underneath
the stage, to the rear of the microscope. It
moves the stage rapidly to position the specimen in relation to the lens. With the coarse
focus knob, the user can focus the lower powers quickly and accurately as well as increase
the clearance of the lens when large objects
are added or removed from the stage. Higher
magnification work requires using the fine
focus knob. On modern microscopes, it is the
smaller-diameter knob that is usually found
concentric to the coarse knob. The fine focus
knob raises and lowers the stage delicately
and becomes the final control when focusing
the image through the oculars and camera.
Frequently, its rim has tick marks that indicate the movement of the stage in microns.
Typically, one full rotation of this control will
move the stage 100 microns, or 0.1 mm.
Above the stage, closest to the specimen, is
the objective lens. Its name reflects its position in the optical path, as it is closest to the
object being examined. The magnification of
each lens is engraved on its barrel as a number
followed by an x. Usually, there are several
objectives mounted on the microscope, and
since they have different powers, they are the
means for varying magnification. They are
mounted on a circular rotating disc called the
nosepiece. To change power, the user rotates
the objective of the appropriate power so that
it lies in line with the eyepiece.
Above the nosepiece is the body of the
microscope, and at its top are the eyepieces. In
a microscope designed for photomicrography,
5
Figure 1-3: A Leitz Ortholux microscope, one of the
first modern microscope designs. This microscope
was made in the 1950s and is suitable for digital
photomicrography. It has a trinocular head that
can hold the full weight of a camera. In older
microscopes of this era, the knobs were separated.
The fine focus knob moves the stage vertically by
only 2 mm, whereas the coarse focus mechanism
can move the stage 20 mm or more.
4
Chapter 1
Basic Microscope Parts
Digital SLR
Camera Adapter Tube
Eyepiece
Trinocular Head
Nose piece
Magnification
Changer Optovar
Objective
Stage
Condenser
Condenser Focus
Field Diaphragm
Focus, Coarse
Focus, Fine
Figure 1-2: A Zeiss WL equipped with a digital SLR
camera. This microscope was manufactured in the
1960s and shows the design changes that made
the microscope more suitable for photography.
The microscope body has a trinocular head with
the vertical tube dedicated to holding the camera:
a microscopist can view a sample through the
eyepieces and conveniently record observations
with the camera. Note how the microscope body
is fixed in position: raising and lowering the stage
accomplishes focus.
There are three functional divisions of the
basic microscope controls: the first is holding the specimen, the second is focusing to
attain a sharp image, and the third is adjusting
the illumination to minimize glare (figures
1-1, 1-2, and 1-3). The stage supports the
specimen, which in turn is mounted on a glass
slide. While appearing simple, the stage is a
precisely engineered flat plate located in the
mid-region of the microscope. Its smooth
surface is perpendicular to the viewing optics,
and it is finely polished to allow minute movements of the slide without any friction or
sticking.
The slide is typically a glass rectangle, 1 mm
thick and 25 mm wide by 75 mm long. It is
made of an optical-grade glass with its surfaces parallel and polished. A sample, which
for many users is a thin slice of tissue that has
been stained with dyes, is placed on its upper
surface. A thin glass cover (coverglass) of a
defined thickness is placed over the sample,
sandwiching it between the slide and the coverglass. The resulting mounted slide is placed
on the stage with the coverglass facing up.
Two clips, one spring-loaded, grip the slide
securely. Two precise mechanical gears move
the clips vertically and horizontally on the
stage to position the specimen for viewing.
The microscopist focuses the specimen by
moving it closer or farther from the viewing
optics. In older microscopes, the slide is fixed
and the viewing optics are raised or lowered
(figure 1-1). On modern research microscopes,
the entire stage assembly is moved while the
optical train is fixed (figures 1-2 and 1-3).
Focusing by moving the stage assembly is
preferred when using a digital camera. Otherwise, the focusing mechanism has to carry
the weight of the camera as well as the microscope head. This additional weight can make it
difficult to turn the coarse focus control. For
the purposes of this book, I assume that the
reader is using a modern microscope with a
focusing stage.
There are two focusing controls: coarse
and fine. Typically, the coarse focus knob is
larger in diameter and is found underneath
the stage, to the rear of the microscope. It
moves the stage rapidly to position the specimen in relation to the lens. With the coarse
focus knob, the user can focus the lower powers quickly and accurately as well as increase
the clearance of the lens when large objects
are added or removed from the stage. Higher
magnification work requires using the fine
focus knob. On modern microscopes, it is the
smaller-diameter knob that is usually found
concentric to the coarse knob. The fine focus
knob raises and lowers the stage delicately
and becomes the final control when focusing
the image through the oculars and camera.
Frequently, its rim has tick marks that indicate the movement of the stage in microns.
Typically, one full rotation of this control will
move the stage 100 microns, or 0.1 mm.
Above the stage, closest to the specimen, is
the objective lens. Its name reflects its position in the optical path, as it is closest to the
object being examined. The magnification of
each lens is engraved on its barrel as a number
followed by an x. Usually, there are several
objectives mounted on the microscope, and
since they have different powers, they are the
means for varying magnification. They are
mounted on a circular rotating disc called the
nosepiece. To change power, the user rotates
the objective of the appropriate power so that
it lies in line with the eyepiece.
Above the nosepiece is the body of the
microscope, and at its top are the eyepieces. In
a microscope designed for photomicrography,
5
Figure 1-3: A Leitz Ortholux microscope, one of the
first modern microscope designs. This microscope
was made in the 1950s and is suitable for digital
photomicrography. It has a trinocular head that
can hold the full weight of a camera. In older
microscopes of this era, the knobs were separated.
The fine focus knob moves the stage vertically by
only 2 mm, whereas the coarse focus mechanism
can move the stage 20 mm or more.
6
Chapter 1
the eyepieces are mounted in a trinocular
head that consists of two eyepieces and a photographic tube. The eyepieces are paired and
angled at 45° and can be adjusted closer or
further apart, thus accommodating individual
variation in the distance between the left and
right eye. To compensate for differences in the
strength of the eyes, one of the eyepieces has
a knurled control that ensures that the degree
of near or farsightedness is equivalent for the
two eyes. The photographic tube is vertical
and serves to hold the digital camera. A slider
on the side of the viewing tube directs the
light to either the eyepieces or the camera.
The eyepieces, also called oculars, have an
engraved magnification number followed
by an x representing the eyepiece’s power.
Together with the objective’s power, the user
can determine the visual magnification of the
microscope. The magnification equals the multiplied value of the eyepiece and the objective.
For example, if the objective is 100x and the
eyepiece is 10x, the total magnification power
is 1000x.
Beneath the stage are the controls and
optics for illuminating the specimen. Almost
all modern research microscopes have a
built-in illuminator that lights the specimen
according to procedures described by Köhler
(see the end of this chapter). Understanding
this procedure is necessary for high-resolution
imaging. Considering that the illuminator is
built in, there is little reason for not using it
correctly.
In the base of the microscope is the field
diaphragm that can be opened or closed with a
large, knurled ring. This controls the expanse
of light illuminating the specimen; a larger
opening lights a larger portion, and a smaller
opening lights a smaller portion. Highercontrast images are obtained by restricting the
Basic Microscope Parts
light to the regions that are being viewed. The
size of this field varies because of the range of
magnifications. Lower magnifications encompass a larger area of the specimen than higher
magnifications. An adjustable field diaphragm
ensures that you can match the range of magnifications to the field of view.
Above the field diaphragm is the condenser,
a group of lenses for focusing the light onto
the specimen. Light is focused by raising or
lowering this assembly, a task accomplished
by a knob that sits beneath the stage and forward of the focus knobs. Additionally, there
is a condenser diaphragm for regulating the
numerical aperture and centering screws for
making the light concentric to the optical axis.
Correct adjustment of the field diaphragm,
condenser focus, condenser diaphragm, and
centering screws are all critical for proper illumination. The following list includes the minimum controls that the user must understand,
starting at the base of the microscope and proceeding up to the eyepieces:
• Field diaphragm - The iris diaphragm
whose diameter controls the area of the
specimen that is illuminated.
• Condenser focus knobs - The knobs that
raise and lower the condenser to concentrate light onto the specimen.
• Condenser centering screws - Two
opposed screws that center the illumination
to the objective lens.
• Condenser diaphragm - The iris diaphragm
located in the condenser that controls the
size of the cone of light projected to the
specimen.
• Coarse focus knob - The knob that raises
and lowers the stage, usually the largest
knob underneath the microscope stage. It
positions the stage rapidly and can be used
to focus objects at low power.
• Fine focus knob - The knob that raises and
lowers the stage for fine focus. Typically one
rotation will move the stage only 0.1 mm.
This knob is smaller in diameter than the
coarse focus knob.
• Stage - The flat plate on which the slide is
mounted. It moves vertically and can be
raised or lowered.
• Mechanical stage - The geared controls that
move the specimen across the surface of the
stage.
• Objective lens - The imaging lens of the
microscope, and the one positioned closest to the object being observed. A number
engraved on the side indicates its magnification.
• Nosepiece - The circular disc that holds multiple objectives and functions as a method
to rapidly change objectives.
• Trinocular head - The three-tube body
above the nosepiece that holds the two eyepieces and the photographic tube for the
camera.
• Eyepiece - The lens closest to the observer’s
eye. It is engraved with its magnification,
and multiplying this value by the number of
the objective gives the total visual magnification.
7
Figure 1-4: Before setting up Köhler illumination, get the
specimen in focus. To do this, place a well-stained slide on the
stage, coverglass facing up. Make sure a 10x objective is in
place and raise the stage with the coarse focus knob until the
slide is just short of the objective. When you look through the
eyepieces, you should see the out-of-focus specimen; if it is
heavily colored with stain, the colors should be evident. Then,
These parts are the essential components of
any modern microscope. Knowing where and
how to use them enables the microscopist to
improve the chances of recording excellent
images. As mentioned earlier, Köhler illumination is required for accurate imaging. The
following section breaks down the process
into manageable steps and provides a baseline for correctly illuminating the specimen.
Failure to follow these procedures can produce
inferior results and potentially damage the
objective lens.
while looking through the eyepieces, turn the coarse focus knob
so the stage goes down, away from the objective. The specimen
should become apparent.
6
Chapter 1
the eyepieces are mounted in a trinocular
head that consists of two eyepieces and a photographic tube. The eyepieces are paired and
angled at 45° and can be adjusted closer or
further apart, thus accommodating individual
variation in the distance between the left and
right eye. To compensate for differences in the
strength of the eyes, one of the eyepieces has
a knurled control that ensures that the degree
of near or farsightedness is equivalent for the
two eyes. The photographic tube is vertical
and serves to hold the digital camera. A slider
on the side of the viewing tube directs the
light to either the eyepieces or the camera.
The eyepieces, also called oculars, have an
engraved magnification number followed
by an x representing the eyepiece’s power.
Together with the objective’s power, the user
can determine the visual magnification of the
microscope. The magnification equals the multiplied value of the eyepiece and the objective.
For example, if the objective is 100x and the
eyepiece is 10x, the total magnification power
is 1000x.
Beneath the stage are the controls and
optics for illuminating the specimen. Almost
all modern research microscopes have a
built-in illuminator that lights the specimen
according to procedures described by Köhler
(see the end of this chapter). Understanding
this procedure is necessary for high-resolution
imaging. Considering that the illuminator is
built in, there is little reason for not using it
correctly.
In the base of the microscope is the field
diaphragm that can be opened or closed with a
large, knurled ring. This controls the expanse
of light illuminating the specimen; a larger
opening lights a larger portion, and a smaller
opening lights a smaller portion. Highercontrast images are obtained by restricting the
Basic Microscope Parts
light to the regions that are being viewed. The
size of this field varies because of the range of
magnifications. Lower magnifications encompass a larger area of the specimen than higher
magnifications. An adjustable field diaphragm
ensures that you can match the range of magnifications to the field of view.
Above the field diaphragm is the condenser,
a group of lenses for focusing the light onto
the specimen. Light is focused by raising or
lowering this assembly, a task accomplished
by a knob that sits beneath the stage and forward of the focus knobs. Additionally, there
is a condenser diaphragm for regulating the
numerical aperture and centering screws for
making the light concentric to the optical axis.
Correct adjustment of the field diaphragm,
condenser focus, condenser diaphragm, and
centering screws are all critical for proper illumination. The following list includes the minimum controls that the user must understand,
starting at the base of the microscope and proceeding up to the eyepieces:
• Field diaphragm - The iris diaphragm
whose diameter controls the area of the
specimen that is illuminated.
• Condenser focus knobs - The knobs that
raise and lower the condenser to concentrate light onto the specimen.
• Condenser centering screws - Two
opposed screws that center the illumination
to the objective lens.
• Condenser diaphragm - The iris diaphragm
located in the condenser that controls the
size of the cone of light projected to the
specimen.
• Coarse focus knob - The knob that raises
and lowers the stage, usually the largest
knob underneath the microscope stage. It
positions the stage rapidly and can be used
to focus objects at low power.
• Fine focus knob - The knob that raises and
lowers the stage for fine focus. Typically one
rotation will move the stage only 0.1 mm.
This knob is smaller in diameter than the
coarse focus knob.
• Stage - The flat plate on which the slide is
mounted. It moves vertically and can be
raised or lowered.
• Mechanical stage - The geared controls that
move the specimen across the surface of the
stage.
• Objective lens - The imaging lens of the
microscope, and the one positioned closest to the object being observed. A number
engraved on the side indicates its magnification.
• Nosepiece - The circular disc that holds multiple objectives and functions as a method
to rapidly change objectives.
• Trinocular head - The three-tube body
above the nosepiece that holds the two eyepieces and the photographic tube for the
camera.
• Eyepiece - The lens closest to the observer’s
eye. It is engraved with its magnification,
and multiplying this value by the number of
the objective gives the total visual magnification.
7
Figure 1-4: Before setting up Köhler illumination, get the
specimen in focus. To do this, place a well-stained slide on the
stage, coverglass facing up. Make sure a 10x objective is in
place and raise the stage with the coarse focus knob until the
slide is just short of the objective. When you look through the
eyepieces, you should see the out-of-focus specimen; if it is
heavily colored with stain, the colors should be evident. Then,
These parts are the essential components of
any modern microscope. Knowing where and
how to use them enables the microscopist to
improve the chances of recording excellent
images. As mentioned earlier, Köhler illumination is required for accurate imaging. The
following section breaks down the process
into manageable steps and provides a baseline for correctly illuminating the specimen.
Failure to follow these procedures can produce
inferior results and potentially damage the
objective lens.
while looking through the eyepieces, turn the coarse focus knob
so the stage goes down, away from the objective. The specimen
should become apparent.
8
Chapter 1
Köhler Illumination
9
Figure 1-5: Lowering
Figure 1-6: When
the stage away from
you close the field
the objective ensures
diaphragm to its
that you won’t
minimum size, you will
inadvertently crash
see a circle of light with
the slide against the
blurred edges. Since
lens. Now adjust the
the condenser is not
fine focus knob until
yet in focus, the edges
the image comes into
of the field diaphragm
sharp focus.
appear fuzzy.
Köhler Illumination
1. Turn on the microscope illuminator and
make sure light is passing through the
field diaphragm.
2. Place a slide (a slide of stained tissue is
good) on the microscope stage. Secure the
slide with the microscope’s clips. Make
sure the coverglass is facing up.
3. Rotate the objective marked 10x into position. It will be perpendicular to the stage.
Make sure it will clear the clips that hold
the slide before rotating it into place.
4. Using the coarse focus knob, move the
stage to just short of the 10x objective.
Be careful not to hit the objective into the
slide (see figure 1-4). You should now be
able to see an out-of-focus specimen.
5. While looking through the eyepieces, turn
the coarse focus knob so that the stage
lowers (moves away from the objective)
to bring the specimen into rough focus.
Lowering the stage ensures that you won’t
raise the slide and hit the objective.
6. Once the image comes into view, adjust
the fine focus knob until the image
appears sharp (see figure 1-5).
7. Close the field diaphragm to its minimum
diameter. You should see a brightly illuminated spot. Its limit is the edges of the
field diaphragm, and generally the edges
will appear blurred (see figure 1-6).
8. Adjust the condenser focus control until
the edges of the field diaphragm become
sharp. When this is accomplished, the
8
Chapter 1
Köhler Illumination
9
Figure 1-5: Lowering
Figure 1-6: When
the stage away from
you close the field
the objective ensures
diaphragm to its
that you won’t
minimum size, you will
inadvertently crash
see a circle of light with
the slide against the
blurred edges. Since
lens. Now adjust the
the condenser is not
fine focus knob until
yet in focus, the edges
the image comes into
of the field diaphragm
sharp focus.
appear fuzzy.
Köhler Illumination
1. Turn on the microscope illuminator and
make sure light is passing through the
field diaphragm.
2. Place a slide (a slide of stained tissue is
good) on the microscope stage. Secure the
slide with the microscope’s clips. Make
sure the coverglass is facing up.
3. Rotate the objective marked 10x into position. It will be perpendicular to the stage.
Make sure it will clear the clips that hold
the slide before rotating it into place.
4. Using the coarse focus knob, move the
stage to just short of the 10x objective.
Be careful not to hit the objective into the
slide (see figure 1-4). You should now be
able to see an out-of-focus specimen.
5. While looking through the eyepieces, turn
the coarse focus knob so that the stage
lowers (moves away from the objective)
to bring the specimen into rough focus.
Lowering the stage ensures that you won’t
raise the slide and hit the objective.
6. Once the image comes into view, adjust
the fine focus knob until the image
appears sharp (see figure 1-5).
7. Close the field diaphragm to its minimum
diameter. You should see a brightly illuminated spot. Its limit is the edges of the
field diaphragm, and generally the edges
will appear blurred (see figure 1-6).
8. Adjust the condenser focus control until
the edges of the field diaphragm become
sharp. When this is accomplished, the
10
Chapter 1
Köhler Illumination
11
Figure 1-8: When
you look through the
eyepiece, the field
diaphragm will have
a circular appearance
with a black limit. Open
the field diaphragm
until its edges just
pass the border seen
through the eyepiece.
This ensures that the
Figure 1-7: Bring
area to be illuminated
the edges of the
matches the area
field diaphragm into
being observed. If
sharp focus by raising
the condenser has
and lowering the
centering screws,
condenser. Typically,
adjust them so that
the circle of light
the field diaphragm
will be at its smallest
is centered on the
diameter when in
circular field of the
focus.
microscope.
image of the field diaphragm is projected
onto the specimen plane (see figure 1-7).
9. Open the field diaphragm until it just
clears the field of view. This ensures that
the region encompassed by the microscope
is fully illuminated (see figure 1-8).
10.If the field diaphragm is not centered in
the microscope eyepiece, use the condenser centering controls to center it.
11.Remove one of the eyepieces and look
down the tube of the microscope. Open
and close the condenser diaphragm,
watching how the circle of light increases
and decreases in diameter. Reopen the
condenser diaphragm and observe the
maximum size of the illuminated circle.
Then, noting this position, close the condenser diaphragm so that the diameter of
the lighted circle is three-fourths that of
the widest field (figure 1-9). For stained
samples, these settings provide the optimum image for revealing fine details while
maintaining contrast. If you close the condenser diaphragm to its minimum diameter, the contrast of the image increases
at the expense of losing fine detail. If you
open it too wide, the image loses contrast
and subtle structures disappear in the
glare.
12.To increase magnification, rotate the
objective with the next highest number
into the optical path. Again, make sure
the objective clears the slide and stage
clips to avoid scrapping and damaging the
objective. Instead of viewing through the
oculars, watch the objective while moving
10
Chapter 1
Köhler Illumination
11
Figure 1-8: When
you look through the
eyepiece, the field
diaphragm will have
a circular appearance
with a black limit. Open
the field diaphragm
until its edges just
pass the border seen
through the eyepiece.
This ensures that the
Figure 1-7: Bring
area to be illuminated
the edges of the
matches the area
field diaphragm into
being observed. If
sharp focus by raising
the condenser has
and lowering the
centering screws,
condenser. Typically,
adjust them so that
the circle of light
the field diaphragm
will be at its smallest
is centered on the
diameter when in
circular field of the
focus.
microscope.
image of the field diaphragm is projected
onto the specimen plane (see figure 1-7).
9. Open the field diaphragm until it just
clears the field of view. This ensures that
the region encompassed by the microscope
is fully illuminated (see figure 1-8).
10.If the field diaphragm is not centered in
the microscope eyepiece, use the condenser centering controls to center it.
11.Remove one of the eyepieces and look
down the tube of the microscope. Open
and close the condenser diaphragm,
watching how the circle of light increases
and decreases in diameter. Reopen the
condenser diaphragm and observe the
maximum size of the illuminated circle.
Then, noting this position, close the condenser diaphragm so that the diameter of
the lighted circle is three-fourths that of
the widest field (figure 1-9). For stained
samples, these settings provide the optimum image for revealing fine details while
maintaining contrast. If you close the condenser diaphragm to its minimum diameter, the contrast of the image increases
at the expense of losing fine detail. If you
open it too wide, the image loses contrast
and subtle structures disappear in the
glare.
12.To increase magnification, rotate the
objective with the next highest number
into the optical path. Again, make sure
the objective clears the slide and stage
clips to avoid scrapping and damaging the
objective. Instead of viewing through the
oculars, watch the objective while moving
12
Chapter 1
Troubleshooting
nosepiece, it will be scratched by the metal
clips of the mechanical stage. For the best
results and to avoid potential damage, stick
with a set of microscope components that are
designed to work together.
Figure 1-9: When the eyepiece is removed,
Troubleshooting
you will see a circle of light, which is the rear of
the objective lens. By opening and closing the
condenser diaphragm, you will see the circle of
light increase and decrease in diameter. Open
the condenser diaphragm until you identify the
Rear
Rearof
ofaperture.
Aperture.Partially
Partiallyfilled
filledwith
withlight
light
from
opened.
Gray is
fromCondenser
condenser Iris
iris 75%
75 percent
opened.
Condenser
iris
occluding
light.
Gray is condenser iris occluding light.
it into position. This will prevent any damage to the objective that would make it
unusable.
13.Repeat operations 6 through 11.
There are two types of objectives: dry objectives that work in air and immersion objectives that work in oil. The operation described
earlier is for dry objectives. Immersion objectives require a minute drop of immersion oil
to be placed on the slide’s coverglass. When
the oil objective is rotated into place, its tip is
immersed in the oil, which joins the top of the
coverglass to the tip of the objective lens (an
initial supply of immersion oil is usually provided by the microscope manufacturer).
Because objectives and eyepieces from different manufacturers often share a common
mount, it is possible to attach lenses from
different manufacturers to a microscope
stand. However, unless you are familiar with
maximum diameter of the light. Then close the
condenser diaphragm until the circle of light is
reduced by one-fourth.
the history of objectives and eyepieces, this
practice is discouraged. Sometimes different manufacturers’ products appear to work
together but have enough differences so that
the resulting images can be inferior or a lens
can be damaged.
In a properly configured system, the objectives will be at a defined height above the slide
so that all of them will be in near focus as you
rotate them into position. The objectives are
said to be parfocal. This is a great convenience
since it allows the user to quickly establish
proper focus after changing powers. All that is
required is a fractional turn of the fine focus
knob.
However, by indiscriminately mixing objectives, one may lose parfocality. At best, this
inconvenience will cause an objective to be
out of focus when it is rotated into the optical path. At worst, the objective will be too
close to the coverglass, and as you rotate the
The information in this chapter should be sufficient to enable someone new to the microscope world to illuminate a specimen properly
for viewing and photography. However, things
don’t always work as planned. The following
list will help you troubleshoot any problems
that may arise:
• Make sure the illuminator is working. Most
lamps have a rheostat for varying light output. Briefly turn it to the maximum value.
If light does not emanate from the field
diaphragm, check to see whether the transformer is plugged into a socket and whether
the socket is live. You can plug a table lamp
into the outlet to make sure it is delivering
juice. If the outlet is active, check to see if
the bulb is good. This may require opening
the microscope and inspecting the tungsten
bulb. A minute break in the filament will
keep a bulb from lighting and you may need
to use a magnifying glass to see the split. In
this case, the solution is to replace the bulb.
If the bulb looks intact, unplug the microscope and determine whether the fuse is
blown. If so, replace the fuse.
• If light is emanating from the field diaphragm, determine whether light is being
blocked at the level of the condenser. If you
raise the condenser, open its diaphragm,
and then open the field diaphragm, you can
inspect the slide to see if light is hitting it.
Note that if the rheostat is set at the highest
value, you should not use the eyepiece—the
light will be too intense to view through the
oculars. If there is no light, check the condenser to see if there is anything blocking
the light from entering it. Frequently, a condenser is equipped with a swing-out holder
for a filter. It is possible for the rim of a filter
to lie directly under the condenser, blocking light from the illuminator. If you see
light rising from the condenser and reaching the slide but you still can’t see the light
through the eyepieces, then light is being
blocked between the objective lens and the
eyepiece. Make sure the objective is properly
centered. When the nosepiece is rotated,
there should be an audible click when a new
objective is placed in the optical path. If the
objective is not properly positioned, it may
block the light. If the objective is in position, the problem may be in the microscope
head. Many trinocular heads have a sliding
prism that can direct the light 100 percent
to the eyepiece or 100 percent to the camera. Make sure the slide is positioned to
direct light to the eyepiece.
• If you have a problem that can’t be solved
by the preceding steps, it’s time to call for
assistance. Some microscopes may have
intermediate tubes or filters that block the
light path. Advanced microscopes have
computer-­controlled shutters. If you are uncertain how to operate them, resist the urge
to start changing the settings randomly.
For a novice, it can be nearly impossible to
reset a microscope after random changes.
In addition, any permanent damage to the
microscope’s components would require significant expense to repair.
13
12
Chapter 1
Troubleshooting
nosepiece, it will be scratched by the metal
clips of the mechanical stage. For the best
results and to avoid potential damage, stick
with a set of microscope components that are
designed to work together.
Figure 1-9: When the eyepiece is removed,
Troubleshooting
you will see a circle of light, which is the rear of
the objective lens. By opening and closing the
condenser diaphragm, you will see the circle of
light increase and decrease in diameter. Open
the condenser diaphragm until you identify the
Rear
Rearof
ofaperture.
Aperture.Partially
Partiallyfilled
filledwith
withlight
light
from
opened.
Gray is
fromCondenser
condenser Iris
iris 75%
75 percent
opened.
Condenser
iris
occluding
light.
Gray is condenser iris occluding light.
it into position. This will prevent any damage to the objective that would make it
unusable.
13.Repeat operations 6 through 11.
There are two types of objectives: dry objectives that work in air and immersion objectives that work in oil. The operation described
earlier is for dry objectives. Immersion objectives require a minute drop of immersion oil
to be placed on the slide’s coverglass. When
the oil objective is rotated into place, its tip is
immersed in the oil, which joins the top of the
coverglass to the tip of the objective lens (an
initial supply of immersion oil is usually provided by the microscope manufacturer).
Because objectives and eyepieces from different manufacturers often share a common
mount, it is possible to attach lenses from
different manufacturers to a microscope
stand. However, unless you are familiar with
maximum diameter of the light. Then close the
condenser diaphragm until the circle of light is
reduced by one-fourth.
the history of objectives and eyepieces, this
practice is discouraged. Sometimes different manufacturers’ products appear to work
together but have enough differences so that
the resulting images can be inferior or a lens
can be damaged.
In a properly configured system, the objectives will be at a defined height above the slide
so that all of them will be in near focus as you
rotate them into position. The objectives are
said to be parfocal. This is a great convenience
since it allows the user to quickly establish
proper focus after changing powers. All that is
required is a fractional turn of the fine focus
knob.
However, by indiscriminately mixing objectives, one may lose parfocality. At best, this
inconvenience will cause an objective to be
out of focus when it is rotated into the optical path. At worst, the objective will be too
close to the coverglass, and as you rotate the
The information in this chapter should be sufficient to enable someone new to the microscope world to illuminate a specimen properly
for viewing and photography. However, things
don’t always work as planned. The following
list will help you troubleshoot any problems
that may arise:
• Make sure the illuminator is working. Most
lamps have a rheostat for varying light output. Briefly turn it to the maximum value.
If light does not emanate from the field
diaphragm, check to see whether the transformer is plugged into a socket and whether
the socket is live. You can plug a table lamp
into the outlet to make sure it is delivering
juice. If the outlet is active, check to see if
the bulb is good. This may require opening
the microscope and inspecting the tungsten
bulb. A minute break in the filament will
keep a bulb from lighting and you may need
to use a magnifying glass to see the split. In
this case, the solution is to replace the bulb.
If the bulb looks intact, unplug the microscope and determine whether the fuse is
blown. If so, replace the fuse.
• If light is emanating from the field diaphragm, determine whether light is being
blocked at the level of the condenser. If you
raise the condenser, open its diaphragm,
and then open the field diaphragm, you can
inspect the slide to see if light is hitting it.
Note that if the rheostat is set at the highest
value, you should not use the eyepiece—the
light will be too intense to view through the
oculars. If there is no light, check the condenser to see if there is anything blocking
the light from entering it. Frequently, a condenser is equipped with a swing-out holder
for a filter. It is possible for the rim of a filter
to lie directly under the condenser, blocking light from the illuminator. If you see
light rising from the condenser and reaching the slide but you still can’t see the light
through the eyepieces, then light is being
blocked between the objective lens and the
eyepiece. Make sure the objective is properly
centered. When the nosepiece is rotated,
there should be an audible click when a new
objective is placed in the optical path. If the
objective is not properly positioned, it may
block the light. If the objective is in position, the problem may be in the microscope
head. Many trinocular heads have a sliding
prism that can direct the light 100 percent
to the eyepiece or 100 percent to the camera. Make sure the slide is positioned to
direct light to the eyepiece.
• If you have a problem that can’t be solved
by the preceding steps, it’s time to call for
assistance. Some microscopes may have
intermediate tubes or filters that block the
light path. Advanced microscopes have
computer-­controlled shutters. If you are uncertain how to operate them, resist the urge
to start changing the settings randomly.
For a novice, it can be nearly impossible to
reset a microscope after random changes.
In addition, any permanent damage to the
microscope’s components would require significant expense to repair.
13
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