Download Microscopy: A Comprehensive Guide to Using the Compound Light Microscope and more Study Guides, Projects, Research Veterinary in PDF only on Docsity!
CHAPTER 2
Microscopy
Mary Ann Seagren
OUTLINE
IDENTIFICATION OF PARTS OF THE MICROSCOPE Oculars (Eyepieces) Objective Lenses Degree of Magnification Parfocal Resolving Power (Resolution) Working Distance Arm and Base Light Source Rheostat for Light Source
Condenser Aperture (Iris) Diaphragm Coarse and Fine Adjustment Stage and Stage Brackets or Clips CARE OF THE MICROSCOPE Putting the Microscope Away EXERCISE 1: LABEL THE PARTS OF THE MICROSCOPE EXERCISE 2: MATCH THE PARTS OF THE MICROSCOPE WITH THE CORRESPONDING DEFINITION
USE OF THE COMPOUND MICROSCOPE Procedure: Use the Microscope to View the Letter “e” Procedure: Calculate Field Size Procedure: Estimate Size of Objects Using a Prepared Blood Film REVIEW QUESTIONS
INTRODUCTION
In veterinary practices and laboratories, the compound light microscope is used by veterinarians and veterinary technicians daily in the course of diagnosing and treating animal diseases. A series of lenses is used to form an image from light passing through the specimen. In this way, the image can be magnified as much as 1000 times. In veterinary practices, compound light microscopes are used for a wide variety of reasons such as:
- To examine the morphology and numbers of blood cells (Figure 2-1),
- To check for the presence of intestinal parasites in feces,
- To examine the contents of urine sediment, and
- To examine an ear swab for the presence of infectious agents such as bacteria, mites, and fungi. The ability to properly use a microscope is essential to ensure the accuracy of these important laboratory tests. In a standard compound light microscope, light from an incandescent source is aimed towards a lens, called the con- denser, which is located beneath the stage (Figure 2-2). The condenser concentrates the light before it passes through a hole in the stage where it then penetrates the specimen. From here the light passes through an objective lens, before being magnified a second time by the ocular or eyepiece. Finally, the light reaches the eye so that what was too small to be seen is now made clearly visible. Some microscopes have a built-in illuminator as shown in Figure 2-2, A, whereas others use a mirror to reflect light from an external source (see Figure 2-2, B ).
24
R
W
P
Figure 2-1 Blood smear. This is a photomicrograph (a photograph of a microscopic field) of a smear of stained canine blood. Note the presence of two white blood cells. Each has a purple multilobed nucleus. They are surrounded by donut-like red blood cells, which have no nuclei. Platelets are seen interspersed among the red blood cells and they appear as pale cell fragments or small dots. W, White blood cells; R, red blood cells; P, platelets. (Photo courtesy Manor College.)
Standard compound
light microscope
A
Monocular
light microscope
Ocular
Objective
Slide
Stage
Condenser
Ocular
Objective Slide
Stage
Condenser
Internal Mirror light source
External light source
B
Figure 2-2 A, Standard compound light microscope. Some microscopes have a built-in illuminator. B, Monocular light microscope. Others use a mirror to reflect light from an external source.
LEARNING OBJECTIVES
In this laboratory, students will learn:
- The location, name, and function of each part of the com- pound microscope, and will learn to use, adjust, and main- tain these parts. 2. To use the microscope to view the letter “e.” 3. To calculate the field size of the various magnifications. 4. To estimate sizes of objects viewed using a stained blood film.
MATERIALS NEEDED
- Compound light microscope
- Prepared microscope slide of newsprint letter “e”
- Prepared microscope slide of a stained blood film
- Prepared slide of a grid, ruled in millimeters (grid slide)
- Immersion oil
- Millimeter ruler
- Lens paper
- Lens cleaner
TO BE IDENTIFIED
Eyepiece or ocular (10× or 5× each) Arm Stage Opening of the stage Fine adjustment knob Coarse adjustment knob Base
Illuminator or light source Light on or off switch Rheostat for light source Iris diaphragm Iris diaphragm lever Condenser Condenser knob
IDENTIFICATION OF PARTS OF THE MICROSCOPE
A labeled photograph of a compound microscope is shown in Figure 2-6. Observe the labeled parts as you read the description and function of each part.
OCULARS (EYEPIECES)
A compound light microscope can either be binocu- lar (containing two eyepieces) or monocular (con- taining one eyepiece). The usual magnification of the eyepiece is 10×. It is important to periodically remove the eyepieces and clean them with lens cleaner and lens paper.
Binocular vision offers greater clarity, detail, and a wider field of view than does monocular vision, but some adjustments to the microscope will need to be made to reap the benefits of binocular vision. First, the distance between pupils (in the eye) varies from person to person, so the distance between the oculars will need to be adjusted to match your interpupillary distance. Second, one or both of the eyepieces may be “a telescoping ocular,” which allows it to be focused. Most people see better in one eye than the other. So the focusing of one or both eyepieces allows the visual acuity in both eyes to be matched to one another. Because our visual acuity is measured in
Body tube
ARM
Stage brackets Control knob of mechanical stage clip
Base
Condenser knob
Coarse focus adjustment knob
Fine focus adjustment knob
Light switch
Oculars or eyepieces
Objective lenses
Lever for iris diaphragm
Light source
A
B
Nosepiece
Opening of the stage
Condenser
Rheostat
Figure 2-6 A and B, The most common microscope used in light microscopy is the compound light microscope. The compound light microscope has at least two magnifying lenses. One is in the eyepiece (ocular), and one is in the objective.
units called diopters, this focusing process is called the diopter adjustment.
OBJECTIVE LENSES
The objective lenses are attached to a revolving nosepiece. The power of each lens is engraved on the side of the objective. The smallest power lens may be 3.2×, 3.5×, or 4×, and is used when first locating and viewing a specimen. Because some specimens can be quite minute and diffi cult to fi nd on a com- paratively large glass slide, this objective is some- times called the “scanning lens.” The next lens is called the low power (LP) objec- tive and is a 10× magnifi cation. It is used for initial, coarse focusing and for the examination of large specimens such as nematode eggs. The high power (HP) objective or the high dry lens offers high magnification without the use of oil. High power lenses may magnify 40, 43, or 45 times, depending upon the objective, and are used for small specimens, such as protists, large yeast, and urine sediments. The highest power lens is in the oil immersion objec- tive. It has a magnification of 97× or 100×. It is used with oil to improve resolution. This lens is used for viewing blood smears and bacterial smears, and to see details (such as cell organelles) in histologic samples.
DEGREE OF MAGNIFICATION
The degree of magnification represents the total amount of magnification that is used to visualize a specimen. It is calculated by multiplying the ocular magnification (usually 10×) with the objective magni- fi cation. For example, if you are using the high power objective (40×) to view a group of cells, the total degree of magnification would be equal to10× × 40 × or 400×.
PARFOCAL
Most microscopes are parfocal, which means that the image will remain focused as you increase mag- nification. This is a characteristic of a good quality microscope. When the microscope is focused under low power, it will remain in focus as you switch to a higher and higher power. It is good practice to begin light microscopy in the lowest power and move up in magnification incrementally, even if the micro- scope is parfocal. This step-by-step approach allows you the opportunity to center the specimen within each new fi eld and to use the fi ne adjustment to perfect the image.
RESOLVING POWER (RESOLUTION)
Resolution is the ability of a microscope to produce a clear image. It is the ability to separate and distin- guish fine details of a specimen. The resolving power is the minimum distance by which two points must be separated and still be perceived as two distinct points. Better microscopes have higher resolution,
that is, objects in the specimen can be closer together and still be seen as distinct from one another.
WORKING DISTANCE
When a specimen is in sharp focus, the working dis- tance is the distance between the objective lens that is in use and the specimen. As stronger lenses are used, the working distance decreases. The specimen and the glass slide on which it rests become closer and closer to the objective as you move from scan- ning to low power to high power. Caution should be taken when using high dry and oil immersion objec- tives in particular, as there is risk of lowering these objectives too far when focusing and jamming the objectives into the glass slide. Some carefully pre- pared slides have been broken this way, not to mention the potential for damaging the lens in the objective. So, be very cautious when focusing with high powered objectives. Observe the working dis- tance when using low power, high power, and oil immersion. The distance should approximate 16 mm, 4 mm, and 1.8 mm, respectively.
ARM AND BASE
The arm connects the base, stage, and body tube. The base is the bottom platform of the microscope, which holds the illuminator. Always be sure to carry a microscope by its arm with one hand while using the other hand to support the base.
LIGHT SOURCE
The light source is usually built into the base. A good light source will have a wide dynamic range to provide high intensity illumination at high magnifications and lower intensities at low magnifications. The best micro- scopes have controls that regulate the intensity and shape of the light beam. If your microscope requires an external light source, make sure the light is positioned so that it strikes the middle of the condenser. The apparent field of an eyepiece is constant regard- less of the level of magnification used. So it follows that as magnification increases, the area of visible specimen decreases. Because you are looking at a smaller area, less light reaches the eye, and the image darkens. With a low power objective you may have to cut down on illumination intensity. When using a high power objective you will need all the light you can get, especially when using a less expensive microscope.
RHEOSTAT FOR LIGHT SOURCE
The rheostat regulates the intensity of light coming from the lamp or light source. To lengthen the life of the bulb, lower the light intensity before turning off the light.
CONDENSER
The condenser is located directly above the light source. It focuses the incoming light into a narrow
Exercise 1: Label the Parts of the Microscope
Below is an illustration of a compound microscope (Figure 2-8). Match the terms with the corresponding parts on the drawing.
Oculars Nosepiece Objective lenses Base Arm
Light source Condenser Iris diaphragm Light switch Rheostat
Coarse adjustment knob Fine adjustment knob Stage Stage adjustment knobs
PUTTING THE MICROSCOPE AWAY
- Move the scanning objective into position and lower the stage as far as possible.
- Always make sure the stage and all of the objec- tives are thoroughly cleaned before putting away the microscope. Remember that oil can damage the lenses. 3. Unplug the illuminator cord and wrap it care- fully around the base of the microscope, being careful of the illuminator. 4. Cover the instrument with a dust jacket when not in use.
Exercise 2: Match the Parts of the Microscope With the Corresponding Definition
USE OF THE COMPOUND MICROSCOPE
PROCEDURE: USE THE MICROSCOPE TO
VIEW THE LETTER “e”
- Carefully unwind the cord from around the base, making sure not to damage any parts of the microscope. Plug in cord.
- Make sure the stage and objectives are as far apart from one another as possible and that the lowest power objective (scanning objec- tive) is in position.
- Clean the oculars and objective with lens paper, if needed.
- Turn on the light source by pushing the light switch to the “on” position.
- Adjust the oculars to match your interpupil- lary distance (distance between your eyes).
- Carefully place the prepared slide of the news- print letter “e” on the stage and secure it in place with stage clips or mechanical brackets, if present.
- Move the slide so that the letter “e” is centered over the stage’s hole.
- While looking from the side, use the coarse adjustment knob to carefully lower the lowest powered objective as close to the slide as pos- sible. Do not allow the objective lens to touch the slide.
- Look through the oculars and use the coarse focus knob to slowly raise the objective away from the slide. When the image becomes clear, switch to the fine adjustment knob to perfect the image.
- Using the condenser knob, raise the condenser so that it is almost touching the bottom of the
microscope slide. If the condenser has select- able options, set it to bright field. Start with the iris (aperture) diaphragm in a closed posi- tion. Slowly move the diaphragm lever so that the aperture slowly opens. You will see increas- ing amounts of light coming through the spec- imen as you move the aperture diaphragm lever to a more open position.
- Take a moment to adjust the oculars. Make a diopter adjustment by adjusting the movable eyepiece(s) so that both of your eyes see through the oculars equally well. Look at the image with each eye individually and then together to confirm balanced vision.
- Calculate the degree of magnification by multi- plying the magnification of the ocular by the magnification of the objective. Enter this figure into the table below. Use the millimeter ruler to measure the working distance from the top of the slide to the bottom of the objective and enter this measurement in millimeters (mm) in the table below (Table 2-1).
_____ Oculars _____ Objective lenses _____ Arm _____ Condenser _____ Iris diaphragm _____ Rheostat _____ Coarse adjustment knob _____ Fine adjustment knob _____ Stage
a. Focuses the image. Can move the stage quickly a large distance. b. Focuses the light into the objectives. Adjustment can change contrast. c. Controls the intensity of the illumination. d. Magnifying lenses that are closest to the speci- men. Can be changed to increase magnification. e. Platform for holding the slide. f. You carry the microscope by this part. g. Focuses the image. Moves the stage in very small increments. h. Controls the diameter of the light beam. i. Magnifying lenses that are closest to your eye. Usual magnification is 10×.
TABLE 2-1 Measurements and Calculations
for Each Magnification
Objective Working Field Power Magnification Distance Diameter 4 × 10 × 40 × 100 × (oil)
- Turn off the microscope light and unplug the illuminator cord. Wrap it carefully around the base of the microscope.
- Cover the microscope with a dust cover.
PROCEDURE: CALCULATE FIELD SIZE
It is useful to be able to estimate the size of objects viewed under the microscope. For instance, the oocyst of Isospora canis looks similar to the nema- tode egg Toxascaris leonina. The major difference between the two is that I. canis is half the size of T. leonina. Being able to estimate the size of both I. canis and T. leonina , therefore, is important in making a correct identification. But how is the size of a specimen calculated? The answer is not calcu- lated directly, but indirectly by first calculating the fi eld size. Field size refers to the amount of a speci- men that is visible through the microscope. As the magnification of a specimen increases , the field size decreases. By knowing the total field size, you can estimate the size of the specimen inside the field.
Here Is How It Is Done....
The diameter of the microscope field and the objects in it are measured in millimeters (abbreviation: mm, 1/1000th of a meter) and micrometers (abbreviation: μm, 1/1,000,000th of a meter). More importantly (and more relevant to microscopy), a micrometer (μm) is 1/1000th of a millimeter (mm) or 0.001 mm. To measure the diameter of a microscopic field, you will need a grid slide. A grid slide is a special microscope slide that contains ruled millimeter squares. Each square is 1 mm by 1 mm. Once a grid slide is acquired, complete the following steps:
- Center the grid slide on the stage of the micro- scope. Using the 4× objective, adjust focus, brightness, and contrast until the image is clear and sharp.
- Move the grid slide until one line of the grid is lined up along the left hand side of the field. Count the number of squares that extend across the diameter of the microscopic fi eld. If the last square is only partially in the field, estimate the fraction of the square that is visible. For example, in Figure 2-9 there are three squares in the diameter of the field and approximately half of the fourth square is showing. So the
length of the diameter of the field is 3.5 mm. Calculate the length of the diameter of your fi eld using the 4× objective and enter your result into Table 2-1.
- Move the 10× objective into position and again calculate the length of the diameter of the field in millimeters. Enter your result in Table 2-1.
- We could keep going and directly measure the length of the high power field (HPF, 40× objec- tive) and oil immersion field (100× objective) as you have been doing. But you can also calculate these fields by using the measurements you already have of the low power field (LPF, 10× objective). Calculating these fields is easier and faster than measuring them directly. Use the following formula to calculate the diameter of the fi elds seen under 40× and 100× objectives. Enter your results in Table 2-1.
Diameter of HPF
diameter of LPF degree of magnification of =
× the LP objective Degree of magnification of the HP objectiive
Example:
What would be the diameter of the HPF if the diameter of the LPF is equal to 2 mm? The degree of magnification of the low power objective is 100 (10× objective × 10 × ocular) and the degree of magnification of the high power objective is 400 (40× objective × 10 × ocular). Therefore:
Diameter of HPF 2 mm 100 = mm m × = = 400
0 5. 500 μ
PROCEDURE: ESTIMATE SIZE OF OBJECTS
USING A PREPARED BLOOD FILM
To estimate the size of objects in the field, simply estimate the percentage of the diameter of the fi eld that is occupied by the object. For example, if a fi eld is 1 mm in diameter and an oocyst takes up half of the field size, then the object is approximately 0.5 mm in diameter. Refer to Figure 2-10 for another example.
Set up for Viewing
Clean the oculars and objective with lens paper as needed. Move the 4× objective into position. Adjust the oculars to your interpupillary distance (distance between your eyes). Carefully place a slide of a blood smear on the stage. If there is no cover slip, be sure that the blood Figure 2-9 3.5-mm microscope field. fi lm is facing upward.
Optimize the Lighting
Turn on the illuminator and adjust it so that the field is bright without hurting your eyes. The higher the magnification, the more light you will need.
Adjust the Condenser
Adjust the condenser using the condenser knob so that the condenser is almost touching the bottom of the microscope slide. This is the ideal setting for viewing stained smears. Start with the iris (aperture) diaphragm stopped down (high contrast). You should see the light that comes up through the specimen increase in brightness as you move the aperture dia- phragm lever to a more open position. Adjust the aperture diaphragm to optimize viewing.
Focus, Locate, and Center the Specimen
Start with the lowest magnification objective lens (4×), to find the specimen and/or the part of the specimen you wish to examine. Start with the speci- men out of focus so that the stage and objective must be brought closer together. Use the coarse focus knob to bring the stage and objective closer together. Once you have found the specimen, adjust contrast and intensity of illumination, and move the slide around on the stage until you have a good area for viewing. Find an area where the cells are close together but not touching. Use the fine focus knob to sharpen the image.
Adjust Eyepiece Focus
Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope focus knob. Move the 10× objective into position over the microscope slide. Turn the fine adjustment to sharpen the image. Locate a specific red blood cell in the field. Refer- ring to Table 2-1, find the field size for the 10× objec- tive that you calculated earlier. Using this figure, estimate the diameter of the red blood cell. Draw the cell and write the diameter in the space provided below.
Figure 2-10 Fecal flotation. This photomicrograph of a fecal flotation specimen shows an egg from the nematode Trichuris. The field size is 140 μm, and the egg takes up half of the field diameter. So we estimate the egg length to be 70 μm. (From Bassert J: Images for veterinary technician educators, Summer 2001. )
10 ×
Diameter of RBC = ____________
Locate a specific white blood cell in the smear and estimate its diameter. Draw the cell and write the diameter in the space provided below.
10 ×
Diameter of WBC = ____________
Turn the 40× objective into position above the micro- scope slide. Use the fine adjustment to sharpen the
Re-examine the stage and lenses to ensure that they are thoroughly cleaned. Turn off the microscope light and unplug the illu-
minator cord. Wrap it carefully around the base of the microscope. Cover the microscope with a dust cover.
REVIEW QUESTIONS
- Why must oil be cleaned off the oil immersion lens before it dries? __________________________________
- Why can’t you use oil with any objective other than the oil immersion objective? _____________________
- How do you calculate the degree of magnification? _________________________________________________
- If you are viewing cells through a 40× objective, what is the degree of magnification at which you are seeing the cells? _______________________________________________________________________________________
- Which focus knob can you use with oil immersion? (fine, coarse, both) _______________________________
- How does the apparent orientation of the letter “e” change when viewed through the microscope? _____
- When you move the slide away from you, in which direction does the letter “e” move? ________________
- As you change to a higher degree of magnification, do you have to decrease or increase the illumination? ____________________________________________________________________________________
- As you move to a higher power objective, does the object become larger or smaller? __________________
- Does the field size become larger or smaller? _______________________________________________________
