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

Learning Objectives List the parts of the microscope Describe the functions of the parts of the microscope Describe the proper use of the fine and coarse adjustment knobs List the steps in examining a microscope slide Discuss the use, care, and maintenance of the microscope Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Key Terms Binocular Compound light microscope Condenser Dark field microscope Fluorescent microscope Numerical aperture Objective lenses Ocular Phase-contrast microscope Planachromatic Resolution Compound microscope is named because it uses a combination of lenses. Ideally, we would want 2 microscopes (one for parasites and one for cytology and hematology)

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 Compound light microscope is used to evaluate blood, urine, semen, exudate and other body fluids. It may also be used to identify internal and external parasites. A dark field microscope is ideal for viewing objects that are unstained, transparent and absorb little or no light. Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

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.

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 x40– 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 Example: something examined under 40x objective through a 10x ocular lens is 400 times larger in diameter than the unmagnified object. (10x ocular lens X 40x objective lens) Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Magnification Total magnification is calculated by: Multiplying the ocular magnification by the objective magnification power Example x10 (ocular lens) x 40x (objective lens) = 400x total magnification Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

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.

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.

The coarse focus adjustment knob The fine focus adjustment knob The coarse adjustment knob located on the arm of the microscope moves the stage up and down to bring the specimen into focus. Fine Adjustment Knob - This knob is inside the coarse adjustment knob and is used to bring the specimen into sharp focus under low power and is used for all focusing when using high power lenses The coarse focus adjustment knob The fine focus adjustment knob Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Substage Condenser Consists of two lenses that focus light from the light source on the object Focused by raising or lowering the condenser Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Aperture Diaphragm Opens and closes to control amount of light illuminating the object Aperture Diaphragm: The purpose of the condenser is to concentrate the light onto the specimen, its diaphragm regulates resolution, contrast and depth of field Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Aperture Diaphragm Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

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 Solvent is a substance, Methanol is the simplest alcohol So be careful using xylene Both Xylene and Methanol are flammable and toxic We use lens cleaner here at the school Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

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!!! Objects could be debris left on the microscope Changing a light bulb requires turning off the power and unplugging the microscope Avoid touching the new bulb directly because oils can shorten the life of the bulb Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

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 (Kem wipes) 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.

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 Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Operating the Microscope (cont.) 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 Do not use the coarse adjustment knob to focus because you will break the slide Remember the coarse adjustment knob lifts the stage Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Operating the Microscope (cont.) 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 Fine adjustment knob focuses Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

Operating the Microscope (cont.) 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.

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 We send the microscopes out for this 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 0.001 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.

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.

Summary Types of microscopes Proper care and use Calibration Digital microscopy