1. Veterinary Practice Laboratory Unit 1
Chapter 3
The Microscope
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

2. Types of Microscopes Compound light microscopes Electron microscopes
Commonly used in veterinary in-house laboratories
Electron microscopes
Research setting or large human medical facilities
Fluorescent microscopes
Phase-contrast microscopes
Reference laboratories
Dark field microscopes
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

3. Compound Light Microscope
Generate image by using a combination of lenses
Optical tube length
Distance between the objective lens and the eyepiece
160 mm in most
Mechanical stage
Holds slide
Coarse and fine focus knobs
Used to focus objects
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

4. Compound Light Microscope (cont.)
Two separate lens systems
Ocular
Located in the eyepiece
Usually x10 magnification
Binocular – two eyepieces
Monocular – one eyepiece
Objective
3-4 objective lenses, each with different magnification
x4 – scanning (not common)
x10 – low power
x4 – high dry
x100 – oil immersion
x50 – low oil immersion (optional)
While looking through the eyepieces, adjust the distance between them so that the two fields appear to be nearly identical and can be viewed as one
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

5. Magnification Total magnification is calculated by:
Multiplying the ocular magnification by the objective magnification power
Example
x10 (ocular lens) x x40 (objective lens) = x400 total magnification
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

6. Eyepieces Mechanical stage Adjusting knobs
A binocular compound light microscope for use in the veterinary clinical laboratory (Courtesy VetLab Supply, Palmetto Bay, FL.)
Eyepieces
Mechanical stage
Adjusting knobs
The microscope head supports the ocular lenses and may be straight or inclined. A microscope with an inclined head has ocular lenses that point back toward the user. This minimizes the need to bend over the microscope to look through the lenses. A binocular head is needed for nearly all routine laboratory evaluations. Trinocular heads are also available and can be used for training purposes or client education. The nosepiece holds the objective lenses. It should always rotate easily and provide ready access to the objective lenses for cleaning.
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

7. When viewed through a compound light microscope, an object appears upside down and reversed. The actual right side of an image is seen as its left side and the actual left side is seen as its right side. Movement of the slide by the mechanical stage also is reversed. Travel knobs are used to move the glass slide and thus the object (or portion of the object). When the stage is moved to the left, the object appears to move to the right.
The mechanical stage controls move the stage back and forth, and left to right
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

8. The coarse focus adjustment knob The fine focus adjustment knob
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

9. Substage Condenser
Consists of two lenses that focus light from the light source on the object
Focused by raising or lowering the condenser
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

10. Aperture Diaphragm
Opens and closes to control amount of light illuminating the object
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

11. Adjusting the Köhler Illumination
1. Secure the slide on the microscope stage
2. Adjust the light source to approximately half of its total brightness
3. Place the x10 ocular lens in position
4. Verify that the eyepiece is at the correct interpupillary distance and that it is focused
5. Focus on the specimen using the coarse adjustment knob
6. Close the field diaphragm and condenser until a small ring of light is visible in the field of view through the specimen
7. If needed, adjust the condenser screws until the light is centered in the field of view
8. Open the diaphragm until the circle of light just touches the edge of the circumference of the field of view
9. Adjust the condenser until the light is in sharp focus. This may make the image darker, so adjust the brightness to compensate
10. Repeat the procedure for each of the ocular objectives
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

12. Care and Maintenance Follow manufacturer guidelines
Only use high quality lens paper to clean lens
Only solvent is methanol or specially designed product
Excessive oil can be removed with xylene
Can dissolve adhesives that secure lens
Wipe clean
Cover when not used
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

13. Care and Maintenance (cont.)
Rotate as you look through them
If objects move with rotation, they need cleaning
Annual cleaning and adjustment by a professional
Extra lightbulbs
Proper location in lab
Protect from excessive heat and humidity
Avoid jarring
Carry with both hands!!!
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

14. Operating the Microscope
1. Lower the stage to its lowest point 2. Turn on the light 3. Inspect the eyepieces, the objective lenses, and the condenser lens, and clean them as necessary (consult the manufacturer’s operating manual for any special cleaning instructions) 4. Place the slide on the stage, with the appropriate side facing up 5. Move the x10 objective lens into position by turning the nosepiece turret (rather than the objective lens)
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

15. Operating the Microscope (cont.)
6. While looking through the eyepieces, adjust the distance between them so that the two fields appear to be nearly identical and can be viewed as one
7. Use the coarse and fine focus knobs to bring the image into focus
8. Adjust the condenser and diaphragm in accordance with the manufacturer’s instructions. This allows one to take full advantage of the microscope’s resolving power
9. When using the x40 (high-dry) objective lens:
• Look for a suitable examination area using the x10 (low-power) objective lens
• Rotate the nosepiece to move the high-dry objective lens into place
• Use the fine adjustment knob to focus on the image
• Do not use oil on the slide when using the high-dry objective lens
• Do not use the coarse adjustment knob to focus on the specimen while the high-power lens is in place
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

16. Operating the Microscope
10. When using the x100 (oil-immersion) objective lens:
• Locate a suitable examination area using the x10 (low-power) objective lens
• Rotate the nosepiece to move the high-power objective lens into place and refocus on the area with the fine adjustment knob
• Rotate the nosepiece so that it is halfway between the high-power and oil-immersion objective lenses
• Place a drop of immersion oil on the slide
• Rotate the nosepiece to bring the oil-immersion lens into place
• Use the fine adjustment knob to focus on the image
• Do not use the coarse adjustment knob to focus on the specimen while the oil-immersion lens is in place
11. When finished:
• Turn the light off
• Lower the stage completely
• Rotate the nosepiece to move the low-power objective lens into place
• Remove the specimen from the stage
• Clean the oil-immersion lens, if necessary
• Cover the microscope
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

17. Calibration Important in identifying objects on the slides
Parasite ova often look similar, but size helps ID them
Should be performed on every microscope in the practice
Stage micrometer
Ocular micrometer
The stage micrometer is a microscope slide etched with a 2-mm line marked in 0.01-mm (10-μm) divisions (Figure 3-7); 1 micrometer (μm) equals mm. The stage micrometer is used only once to calibrate the objectives of the microscope. After the ocular micrometer within the compound microscope has been calibrated at x4, x10, and x40, it is calibrated for the service life of the microscope; the stage micrometer is never used again. The stage micrometer should therefore be borrowed from a university or other diagnostic laboratory rather than purchased.
The ocular micrometer is a glass disk that fits into one of the microscope eyepieces. It is sometimes referred to as a reticle. The disks impose an image of a net, scale, or crosshairs over the viewing area. The reticle should be mounted in a separate ocular lens that can be removed and replaced with a nonreticle assembly for times when the scale is not needed. The disk is etched with 30 hatch marks that are spaced at equal intervals. The number of hatch marks on the disk may vary, but the calibration procedure does not change. The stage micrometer is used to determine the distance in micrometers between the hatch marks on the ocular micrometer for each objective lens of the microscope being calibrated. This information is recorded and labeled on the base of the microscope for future reference.
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

18. Digital Microscopy Use optics and a camera to capture an image
Photomicrographs can be added to patient files
Becoming more affordable
Evaluate cost, resolution, and quality of images
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

19. Summary Types of microscopes Proper care and use Calibration
Digital microscopy
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.