Cytology Lab

Microscopy and Cell Organelles

Objectives: To learn how to use and adjust your microscope for optimum resolution and contrast and to study cell organelles in tissue sections.

Reading: RR&K: Chapters 1-3.

Figure 1.9 and page 17 are particularly useful for understanding the principles of the microscope and its alignment I. Microscopy A. Introduction At the beginning of the laboratory, you will be issued a microscope and two slide boxes. You will use this microscope and these two slide boxes throughout the term. Your microscope will be identified by the last 6 digits of the number above the bar code on the sticker. You are responsible for the condition of this microscope. Please treat it as if it was your own. It is a delicate instrument that will enable you to view and appreciate the microscopic world of cells and subcellular structures. B. Slide boxes There are 28 sets of slide boxes for the laboratory, 24 in the Histology Laboratory itself, 2 in the PIMS Learning Center, 1 in the Study Room in Conradi Building and 1 in the instructors office. Sign the card that is pasted on the cover of the slide box. This will enable users in the other two lab periods to know with whom they share the box.

For the most part, every slide box has at least one example of every tissue and organ but not all boxes have exactly the same slides. There are sometimes as many as 5 different examples for each numbered slide. This is a mixed blessing. On one hand, it gives you access to more examples of the tissue and organ than you would normally have. On the other hand, some slides are simply not as good as others. When there are multiple examples of one slide you are free to swap with your neighbor. Make a BIG effort to get your original slides back and to put them back in their proper slot so that the next person does not have to hunt for the slide.

In those instances where there are insufficient slides to fill all slide boxes, please share these with your neighbors. Keep tract of these slides and return them to their original box at the end of the lab period.

C. Setting up the Microscope Before plugging in the power cord, for safety reasons, please turn the microscope off. The OFF/ON switch (unlabeled) is located in the bottom front on the lower left side. The microscope is OFF is when the button is depressed on the left hand side and ON is the button when depressed on the right side. Plug the power cord into the mains. There is a dial on the lower right side of the microscope that can be used to adjust the illumination intensity.

The key features of the microscope are the field diaphragm, the condenser lens assembly, the objectives, the rotating nosepiece, condenser iris diaphragm, objective focusing knob, eyepiece.

a. field diaphragm - The field diaphragm is used to restrict the area of illumination. This is located on the microscope base. The field diaphragm prevents stray light from degrading the image contrast.

b. condenser lens assembly - Used to focus the illumination onto the slide. It is located just below the specimen stage. There are two knurled stainless steel screws, one on each side, 90° to each other, that are used to center the field diaphragm within the field of view.

c. objectives - There are 4 objectives of magnification 4x, 10x, 40x, 100x. They form the primary image of the specimen, which is then magnified by the eyepiece to give the total image magnification. The 4x, 10x and 40x lenses should never be used with immersion oil. The 100X lens is an oil immersion lens. Do not use it without immersion oil. The immersion oil should be wiped off the lens with lens tissue only.

d. rotating nosepiece - Allows convenient selection of the appropriate objective. Get used to rotating the nosepiece clock-wise, which is in the direction from low to high magnification. In this way, you will not accidentally brake slides and can avoid getting oil on your 40x objective.

e. condenser iris diaphragm - Restricts the angle of illumination on the specimen and thereby alters the resolution and the depth of focus. It is a black lever located on the condenser lens assembly at the front just above the Olympus/Japan label.

g. objective focusing knob - Focuses the specimen. There is one on both sides of the microscope. The outer knob is for coarse focusing, the inner knob is for fine focusing. Find and focus the specimen first at low magnification by using the coarse focusing knob. Use the fine focusing knob only when switching from low to higher magnifications.

The inner focusing knob on the right hand side is calibrated and can thereby be used to measure the depth of features in the section and the section thickness. There is a line on the coarse focusing knob that is used as a reference when measuring thickness.

h. eyepiece - These are 10x lenses that form the final image. The inter-ocular distance can be adjusted to your individual needs and one of the eyepieces can be focused independently of the other.

i. stage assembly – Two knurled knobs, one above the other control the X and Y movements of the stage. A small clip located on the top right side holds the slide in place.

D. Microscope Alignment

The condenser’s focus. During normal operation, the condenser is not changed, but it is wise to check the condenser focus at the beginning of the laboratory period because it is easily knocked out of alignment. It is also quite common to accidentally grab the condenser and attempt to focus the image with it. If you do this, you will have to refocus the condenser. The slang term for focusing the condenser is "racking" (the focus moves on a rack and pinion) and "racking down" the condenser means lowering it. You do not want to be caught with your condenser "racked down". Closing the condenser diaphragm is referred to as "stopping down the condenser".

Your microscope is equipped with Koehler illumination. There is a simple, easy and precisely defined method for aligning such instruments.

(a) Focus on any well-stained slide with the 10X objective. Have the condenser diaphragm opened all the way. After focusing the image, close the field-stop diaphragm all the way.

(b) Adjust the condenser’s focus until the image of this diaphragm is sharpest. The colored fringes around this image arise from the chromatic aberrations of the condenser lenses.

(c) Adjust the two field-diaphragm centering screws to center the image of the diaphragm. (Be careful not to loosen the screw on the condenser or it will fall out. The centering screws are on the field lens.)

(d) Switch to the 40X objective and repeat this adjustment. You will find that the image of the diaphragm appears very fuzzy at this magnification, and it can’t really be sharply focused. Close the condenser diaphragm and the image of the field diaphragm will sharpen. Focus the condenser and readjust the centering screws if necessary. Once the condenser is focused for the 40X lens, it should be pretty good for the lower powers. The image of the field diaphragm is too fuzzy to be focused with the 100X objective so the 40X alignment is the final alignment.

(e) Refocus the image with the 4X objective and spread the field stop diaphragm until it just fills the field of view. The best illumination for any given objective is obtained by setting the field diaphragm to just fill the field of view. However, this will be tedious during the laboratory periods so the 4X position will be the best for general work. If you feel you are having trouble with this alignment, open the field stop diaphragm all the way and leave it there.

The condenser diaphragm controls the numerical aperture (N.A.) of the condenser. For the best resolution and for sharpest focus (minimum depth of field) the condenser aperture should be equal to the objective aperture (in practice it is usually made slightly smaller). Image contrast and depth of focus can be enhanced by closing the condenser diaphragm, which makes the aperture of the condenser much smaller than that of the objective. This is at a loss of resolution. For critical work, each time the objective is changed, the condenser diaphragm should be adjusted. The following procedure can be used for this adjustment:

(a) Focus your slide with the 40X objective and remove one eyepiece. What you see when looking down the tube is the back focal plane (bfp) of the objective.

(b) Watch the bfp as you open and close the condenser diaphragm. With the diaphragm closed, there is a small circle of white light surrounded by a larger circle of dim colored light. Move the specimen away from the field and note that the colored light disappears. The colored light has been scattered or diffracted by the specimen. The small circle of white light has passed through the specimen without scattering. The large circle of diffracted light is limited by the numerical aperture of the objective. The circle of white light is limited by the condenser diaphragm and shows the N.A. of the condenser. If you open the condenser diaphragm the circle of white unscattered light should fill the whole bfp. Under this condition, the N.A. of the condenser is equal to the N.A. of the objective.

(c) Ideally, the N.A. of the condenser should be 2/3 of the objective. This is achieved by closing the condenser diaphragm until the circle of white light just cuts into the bfp of the objective. Since the N.A. of each objective is different, the condenser diaphragm should ideally be readjusted each time the objective is changed.

(d) Switch to the 10X objective and notice that the condenser diaphragm must be closed much further to cut into the bfp of this lens. What does that tell you about the relative N.A. of the 40X and 10X objectives? Look on the lenses themselves (do not remove them to do this!) to read out the N.A. With the 100X lens, even with the condenser diaphragm wide open the bfp will not be filled. The N.A. of the condenser is about 1.0. What is the N.A. of the 100X objective?

(e) The quickest and easiest way to adjust the condenser diaphragm is to open it fully and while looking at the specimen (ocular in place), close the diaphragm slowly until you just notice a change in image quality (brightness and contrast). [When you look at the bfp, you will notice that it is about 2/3 full].

Having said all that, you may find it simplest to work with the condenser diaphragm fully open. You will probably not notice the difference in image quality. However, you should be familiar with this procedure because you will be sharing the microscope with students in other laboratories and the instrument alignment may be quite different from one lab period to the next.

E. Use of the oil immersion lens Most of the sections in the Histology collection are thick (10-14 µm and thicker) paraffin sections and can be observed using only the 4X, 10X, and 40X objectives. The oil immersion lens is only useful for blood smear slides and thin, plastic embedded sections (1-2 µm) and of which there are precious few in the collections. Some of the thinner paraffin sections may reveal cell organelles "under oil" so you should become familiar with the use of oil. Those sections that will reveal fine detail under oil will be identified in subsequent laboratory periods.

The first step in examining a slide under oil is to examine it thoroughly with the lower power objectives. It is almost impossible to scan a slide under oil. When you find an area of interest, focus it with the 40X objective. Next rotate the nose piece midway between the 40X and the 100X objective and apply a small drop of oil to the slide. Slowly rotate the 100X lens into position. With the image in focus at 40X the 100X lens should move smoothly into the oil drop without touching the slide. Focus carefully using only the fine focus. The 100X oil immersion objective reveals specimen detail near the theoretical limit of the light microscope.

Be very careful not to move the 40X lens into position if you have oil on the slide - you will get oil on the lens and it is very difficult to clean. Ask you instructor if this misfortune should befall you. You can, however, return to 4X and 10X, and it is useful to do this frequently to regain the perspective of the large field of view at low power.

J 12 Helpful Hints for Slide Observation (1) Ask your instructor for help if you feel there is any problem with your microscope which you cannot handle. You are responsible for damage to the microscope. Under no circumstances should you try to dismantle the scope.

(3) When you place the slide on the microscope, the image will be rotated 180° relative to its orientation as seen by eye.

(4) Be sure that slides are placed on the stage with the cover class (specimen side) up. If you cannot get your specimen in focus at the higher magnifications, then it is probably upside down.

(5) Always start examination with the 4X objective and rotate the nose piece clockwise to select higher power objectives. This will prevent you from passing the 40X objective through the drop of oil left behind after oil immersion examination.

(6) Scrupulously avoid contacting any lens surface with fingers or other foreign objects.

(7) A very dirty objective can cause glare and fogging in the image, but a small amount of dirt will not be noticed. More harm can be done by unnecessary cleaning than from a small amount of dirt so it is best too leave the objectives alone.

(8) It is not necessary to clean the oil immersion lens after each use, but at the end of the day the excess oil should be gently wiped away with a lens paper.

(9) Oil should be cleaned off the slides by wiping them with the gauze cloth or lens tissue. Xylene is not necessary.

(10) Get used to examining the slide with the naked eye before mounting it on the microscope. This will help orient you with respect to the gRR&Ker features of the specimen.

Get used to looking at the whole slide under low power to get a sense of the different structures that are present:

Scan all margins of the tissue.
Find the cut and natural surfaces/edges; identify lumen, if present.
Look for the epithelial lining/covering the surface on the.
Don’t base your identification on the first structure you see when another structure that you haven’t bothered with might change your diagnosis.

(12) When observing specimens, make it a habit always to start with a lower power and work up. DO NOT start looking at a new specimen or a fresh area with the 40X or 100X lens. Focus first at low power, then switch to the higher power. Be careful: objectives are usually parfocal (i.e. in focus at the same specimen height), but yours may not be. Do not embarrass yourself by ramming the objective into a slide.

II. Cell Organelles

A. Purpose: Identification of specific cell organelles. These cell organelles will be important in unknown slide identification. There are 3 specific cell structures that we will examine in this lab exercise. 1. Nucleus/ chromatin/ nucleolus

2. Cell boundaries/ nuclear boundaries

3. Cytoplasmic structures/ secretory granules

B. Liver - There are several groups of slides that can be used for this exercise.
105A,B Liver, Pig, MA
106A,B Liver, Human, H&E

Porcine (pig) liver Human liver Examine first the pig liver slide. A characteristic feature of liver is the uniform appearance of the cells and their arrangement in cords (rows) of cells. Some of the pig liver slides (105a) are stained with Mallory-Azan stain which stains collagenous connective tissue blue and cellular components red. Red Blood Cells (RBCs) are poorly stained with this method and appear as polygonal shaped "ghosts". The other pig liver slides (105b) are stained with H&E which does not contrast connective tissue as well as Mallory-Azan. However, you can still see the connective tissue outlines (interstitium) because their structure differs from that of the parenchymal cells.

Examine the slide with 4X. You will see several blue polygonal outlines on the Mallory-Azan stained slide, which represent the classic liver lobules, the functional unit of the liver. In the center is a structure known as the central vein, which at higher power will appear filled with clear-staining polygonal RBCs. In between the blue outlines and the central vein are cords of cells radiating out from the central vein. Make a sketch of the liver lobule. Examine these cords of cells at higher power. Pig liver is unusual in that the connective tissue surrounding the lobules is prominent.

At 40X, the cords are separated by pale staining regions (lack of staining), which are the venous sinuses lined by endothelial cells, like any other vessels. The cuboidal cells in the cords of the liver are named hepatocytes, comprising the parenchyme of the liver. Their cell boundaries should be distinct as well as their nuclei. In many nuclei you will be able to distinguish a dark spot. What do you think this spot represents ? Sketch the cords of cells and their nuclei. Check Fig. 17.1 (page 498) for the 3-D arrangement.

The human liver slide does not show the prominent oulines of the liver lobule that were present in the pig liver. You should nevertheless still be able to find the central vein and the cords of hepatocytes. Because H&E stains nuclei a dark blue, you will be able to distinguish different shaped nuclei. The large pale ones are the hepatocytes. Elongated nuclei are endothelial cells, which line the liver sinuses.

107 Rabbit liver & gall bladder, PAS&H stained
If you have time look at this slide. Per-iodic Acid Schiff (PAS) reagent specifically stains carbohydrate which is thereby colored an intense red. The Hematoxylin counterstain shows the cell nuclei. Note that the red glycogen granules are not uniformly distributed among the parenchymal cells nor are they spread uniformly within the cells. This slide is an example of specific staining procedures, which can be used to show to best advantage particular features of a cell or tissue. You will experience numerous examples of specific staining during the course.

C. Pancreatic Acinar Cells - The slide boxes contain many different pancreas slides with different stains. Probably the best is 112c.
112A,B,C - Pancreas
113A,B,C,D - Pancreas
114 Pancreas head, H&E

Slide 112C is the best of the pancreas slides for visualizing the secretory granules. The parenchymal cells of the pancreas are arranged in rounded clusters called acini (Latin, acinus= berry or grape). The nuclei are round and located in the basal part of the cell, stained blue by Hematoxylin. The secretory granules are apical (L: apex=peak) stained pink to red in by eosin H&E stained sections. The secretory granules contain digestive enzymes. A small duct at the apex of the acinus conducts the secretory product to larger ducts and eventually to the duodenum. Sketch a typical acinus and the acinar cells. Can you identify any other cellular groupings within the parenchymal tissue? The basal part of the acinar cells, stained blue in 112c, is the rough endoplasmic reticulum. Early histologists named it ergastoplasm.

D. Spinal cord and dorsal root (sensory) ganglion cells.

38A,B Spinal cord and spinal ganglion.
39 Nissl bodies, neurons

Spinal cord Dorsal root ganglion
There are 2 versions of slide 38a. Both have the spinal cord but some have very little ganglion associated with it. Examine this slide at low power. The dark staining, butterfly shaped structure in the center is the gray matter of the spinal cord. Check plate 44 on page 301 of your text for a diagram. You will find neurons in the gray matter. Ignore the pale staining regions which are the white matter which contains axons and glial cells. Neurons are exceptionally large cells with pale, vesicle-like nuclei and very prominent nucleoli. Note that the nucleoli of the neurons are as large as the nuclei of the glial cells in the surrounding matrix. The prominent cytoplasmic staining (slide 39) is referred to as "Nissl substance". Note that the Nissl substance is found in the nerve cell body and dendrites but not in the axons.

Slide 38B shows neurons stained with H&E revealing their large size and granular cytoplasm

Slide 39 is stained specifically for Nissl substance. You will be able to verify that the Nissl substance does not extend into the axon. This slide is stained with only a basophilic dye (crezyl violet), thus acidophilic components of the cell are unstained. There are not enough examples of this slide to go around so share with your neighbor.

Appendix I - Table of Histological Stains Used in this Course

Name/ Used mostly to stain	Chemical nature of dye	RESULTS
Hematoxylin/ General nucleal	Basic	Stains acidic ("basophilic") structures (chromatin, proteins) deep blue
Eosin/ General cytoplasmic stain	Acid	Stains substances with high pH to various shades of red/pink ("eosinophilic").
Toluidine Blue/ General stain for plastic-embedded semi-thin sections	Acidic	Stains mucin reddish-violet; metachromatic stain for granules of mast cells which are stained reddish-violet
Per-iodic Acid Schiff (PAS)/ Any tissue for glycocalyx and glycogen	Aldehyde	Stains glycogen, starch, cellulose red.
Iron-hematoxylin/ Any tissue for subcellular organelles	Basic	Stains nuclear substance, chromosomes, mitochondria, centrioles, muscle striations blue to black
Basic Fuschin Glandular tissues	Basic	Stains elastin deep purple; mast cells, chief cells of gastric mucosa, beta cells of pancreas, basophils of pituitary and some kinds of mucin-purple
Mucicarmine Mucinous glands	Basic	Stains mucin red
Masson’s Connective tissue	Combined staining	Stains chromatin brown-black; nuclei-red; zymogen granules-purple; cytoplasmic elements-red to mauve; collagen, mucous and connective tissue-green
Mallory-Azan Connective tissue	Azocarmin (basic) Anillin blue (acid)	Stains nuclei red; muscle & some cytoplasmic elements red to orange; dense cellular tissue-pink collagen dark blue; connective tissue and hyaline cartilage blue;
Verhoeff’s Elastic fibers in connective tissue	Combined stain	Stains elastic fibers-blue to black; nuclei-blue to brownish black; collagen-red; cytoplasmic elements yellow.
Wilder’s Reticular fibers of basement membranes and lymphatic tissue	Silver impregnation	Stains reticular fibers black
Fat stains Adipose tissue	Lipophilic	Different dyes (Oil red, Congo red, Sudan black) can be used on non-embedded sections (tissue sectioned when frozen, cover-slipped without using organic solvents).
Wright’s Blood smears	Combined stain	RBCs stained yellowish red; Polymorphonclears (neutrophil granulocytes): nuclei-dark purple; granules reddish lilac; cytoplasm-pale pink. Eosinophile granulocytes: nuclei-blue; granules orange-red; cytoplasm blue. Basophile glanulocytes: nucleus purple to dark blue; granules-dark blue. Lymphocytes: nuclei-dark purple; cytoplasm-blue. Platelets: granules-violet to purple
GIEMSA Blood smears	Combined stain	Stains nuclei of leukocytes-reddish purple; otherwise similar to Wright’s

Klüver-Barrera
General stain for the central nervous system
Cresyl violet (acidic)
Luxol fast blue (LFB)
Cresyl violet alone = Nissl strain
Stains rough endoplasmic reticulum (Nissle substance) violet
Stains myelin blue (green); heterochromatin blue

Golgi
Impregnation for nervous tissue
Silver impregnation Selectively impregnates SOME neurons with entire dendritic arborization. Impregnated neurons are brown to black, neuroglia black, background orange-brown.

Cajal
Impregnation for nervous tissue

Silver impregnation Neurons are brown, nuclei unstained, nucleoli and neuronal processes are black