Microscopy ...the embiggening of the brain
There are three types of microscope available in the lab for
examining your slides: The Bausch & Lomb Microprojector,
The Wild macroscope, and the Zeiss compound light microscope with epifluorescence option. This document is not designed to
serve as a comprehensive instruction set on any of these microscopes,
rather it is intended to provide a few tips to those who have a nodding
acquaintance with microscopes.
The Microprojector
Microprojectors are used to scan through your
microscope slides and make paper tracings of critical sections. The operation
of the microprojector is self evident.
- Magnification is a function
of the objective selected and the distance that the image is projected. An
estimate of the magnification can be obtained by projecting a transparent
ruler under the same conditions that the microscope slide was projected.
- Coverslipped
side of the slide should go down. Coverslips
always face the objective.
- When microprojector
bulbs were discontinued, the microprojectors
were converted to use halogen bulbs. These bulbs cause much more fading of
stained slides. Avoid long exposure whenever possible.
- Some of the microprojectors are fitted with a mirror to allow
projection onto a wall for teaching purposes. The head of the microprojector can also be tipped back to do this, but
it is less convenient.
![](scope1.jpg)
Macroscope
The macroscope is used for low power examination
of sections and low power photography. The basic operation of the scope is
straight forward. Turn on the light, place the slide under the macrozoom objective and focus.
- Adjust the interpupillary distance to suit your eyes, by gently
increasing or decreasing the distance between the eye pieces until you
have a single circular field of view.
- The little knob beneath the
eye pieces (16) is the control knob for the aperture diaphragm. The higher
the aperture, the better the resolution. The depth of field, however,
decreases with increasing aperture. At the left hand mark on the scale,
the aperture is fully open.
- There is an additional 2.0X objective
screwed in to the end of the macrozoom
objective. Removing this objective will increase the field of view and
increase the working distance from 42 to 102 mm.
- The knob to the left of the
eyepieces controls the beam splitting prism which directs the image to the
eyepieces(VIS 100%), the
camera(PHOT 100%), or both(PHOT 50% VIS 50%).
Macroscope Photography
Loading
the Camera
- Insure lever(49) points to the white spot.
- Push catch(44) left, open cover.
- Pull out rewind knob(46),insert film, and push rewind knob back in.
- Turn film transport drum(47) to the left using the teeth until the take-up
spool(48) and film transport knob(50) move with it.
- Depress transport
release lever(49). Turn film transport knob(50) CCW until the slit on the take-up spool faces
up.
- Pull the film over the
transport drum(47) and insert the end into the
slit of the take-up spool.
- Turn film transport knob(50) CCW until stop is reached. Depress transport
release lever(49) and advance film 1 frame. Drum(47) must engage both pines of perforation on the
film.
- Close cover.
- Set the line between
35 & 1 on the automatic film counter.
- Advance film by 2 more
frames, first pressing the transport release lever(49)
each time. Film counter should point to 1.
- Removing Film
- Swing transport
release lever(49) to vertical position.
- Unfold crank of rewind
knob(46) and turn clockwise until there is no
resistance to turning.
- Fold up crank and
switch transport release lever(49) to white
spot.
- Push catch(44) left to open magazine cover.
- Pull out the rewind knob(46) and remove film cartridge.
- Dioptric
Correction
- Without introducing a
slide, illuminate the field of view.
- With the eyepiece with
graticule
- Without looking into
it, turn the lens of the eyepiece to "+5".
- Look through the eyepiece
and turn the lens slowly towards "-5" until the vertical of
the crosshair is clearly seen as a double line.
- Select the highest
magnification.
- Focus on a flat
object (cross marked on paper) with the focusing drive.
- With the eyepiece without
graticule
- Set to the lowest
magnification.
- Without looking into
it, turn the lens of the eyepiece to "+5".
- Turn the lens slowly
towards "-5" until you see the object in sharp focus.
- At the highest magnification,
use both eyes to inspect image sharpness. If necessary refocus with focussing drive, but do not redjust
eyepieces.
- Taking Pictures
- Turn on Photoautomat.
- Enter film speed using
ASA/DIN key.
- Press MODE key
until "AUTO" LED is lit.
- Focus on slide and
move slide until your object if framed well in the graticule.
- Using the beam
splitting prism control knob, send the light to the camera (PHOT 100%).
- Depress the EXPO
key.
The Compound Microscope
The compound microscope is used for higher magnification examination of
slides. This microscope is outfitted for epifluorescence
microscopy as well as transmitted light photography. There is a camera mounted
on this microscope. Proper operation of this microscope is a skill that must be
beaten in to you and is, therefore, beyond the scope of this document. This
document will provide a few tips, but is not a substitute for proper
instruction.
- Kohler Illumination
In order to get the best image from the microscope, it is critical that
the light path be set up properly. The method for doing this is called Kohler
Illumination..
Proceed as follows:
- Turn on the
microscope and place a microscope slide on the stage.
- Using the coarse and
fine focus knobs, focus on the tissue.
- Close down the iris
of the field diaphragm at the base of the microscope.
- Slowly start lowering the condenser using the condenser focusing
knob until the edges of the diaphragm are in sharp focus along
with the tissue.
- If the iris is not centered
in the field of view, center it using the two screws located near the
back on each side of the condenser carrier.
- When the light is
centered, close down the iris aperture on the condenser until the area
outside the light field is evenly dark.
- Open up the iris of
the field diaphragm until the edges of the iris are just out of view.
To take measurements of objects in your field of
view, such as cell bodies or pipette tips you need an eyepiece with a built in graticule. The graricule must be
calibrated for each objective that you use. Proceed as follows:
- Place a stage
micrometer in the slide holder on the stage.
- Using the left/right
and anterior/posterior specimen moving knobs,move the stage micrometer until the ruler is
in the center of the field of view. A typical micrometer might have a 1
mm ruler marked off in 5 or 10 µm divisions.
- Insure that the
micrometer is in sharp focus.
- Turn the eyepiece
with the graticule until the graticule overlaps the stage micrometer.
- Use the micrometer to
calibrate your eyepiece graticule. Example:
If it takes 80 divisions in the eyepiece graticule
to cover 1 mm of the micrometer, then each division on the graticule is 1/80 mm or 12.5 µm.
- Photography
- Focus on your slide
in the normal way.
- Center your picture
in the inner rectangle of the eyepiece graticule.
- Set the film speed
(the ASA number is found on the film pack). Using the large dial on the
left of the MC63 unit, turn the dial until film speed lines up with the
small rectangle located at about 1:30
to the dial.
- Set the large dial at
the right to "AUTO".
- Pull out the knob
located to the right of the eyepieces to allow light to travel to camera.
- Press "EXP"
button.
The lab has recently acquired a
digital camera. A microscope eyepiece was machined to screw into the camera in
the same way an optional camera lens would. The camera can now be used to take
pictures through the microscope without an expensive c-mount adaptor by simply
sliding the camera into one of the eyepiece tubes.
In the image at the right you can see the camera in operation. The image of the
specimen appears on the camera display.
The image can also be routed to a TV monitor as seen in the image below. This
makes the camera an excellent teaching tool.
When taking photos, you can gain greater control of the picture by setting the
camera in manual mode.
![](nikon-tv.jpg)
Steve Milway
Last
modified: Monday, December 22, 2003