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Source: http://www.doksinet How to Approach Histology Images Welcome to the fun and fabulous world of Histology! It is our hope that upon completion of this introduction to image interpretation you will be able to confidently approach histology images or slides and that you will not have to resort to memorization to pass the histology (or pathology) portion of your examinations. Let us begin When confronted with an image on an exam or computer screen, you should first determine the plane of section (way in which the specimen was “cut,”) – i.e longitudinal, horizontal/transverse or oblique For example, the images shown below are of skeletal muscle. The image on the left shows a crosssectional plane of section while the image on the right shows a longitudinal plane of section. Source: http://www.doksinet It is quite possible that you will be presented with images that show a mixture of the two, as seen below in this slide of smooth muscle. Source: http://www.doksinet Second,

if a magnification bar is not provided you should determine if you are looking at a low (lots of cells), medium or high magnification (very few cells) image. The following high magnification (left) and low magnification (right) images are all slides from a lung specimen. Source: http://www.doksinet Third, it will be impossible to identify every cell type that you see in many slides. Please keep this in mind as you view images It will prevent unnecessary frustration. LIGHT MICROSCOPY Stains and Staining Techniques All of the images shown above and the majority of the images that you will see on exams will be light microscope images. Interpretation of light microscope slides will involve the steps discussed above but knowledge of the stains used is extremely helpful, if not, essential. The most commonly used stain in light microscopy (LM) is Hematoxylin and Eosin (H & E). Hematoxylin is a basic dye (positively charged) that stains the Source: http://www.doksinet

negatively-charged portions of DNA and RNA purple or dark blue. In contrast, Eosin is an acidic dye (negatively charged) that stains positivelycharged components such as proteins pink. Other dyes and staining techniques are used based on the organelle or component of interest. Another commonly used staining technique is known as immunohistochemistry. In immunohistochemistry, an antibody against a tissue, protein, etc. is covalently linked to an indicator The antibodyindicator complex is added to a piece of tissue on a glass slide, allowed to bind and unbound antibody is washed off (a). Next, substrate that is normally colorless is added to the mixture (b). Ideally, the interaction between the indicator and the substrate results in the production of a colored product which is visible using LM(c).1 1 Wheatre’s Functional Histology p. 407 Source: http://www.doksinet The final product looks like the following: A special type of immunohistochemistry, immunofluorescence, is often

used in nephrology (study of the kidneys). Although the basic steps are similar, in immunofluorescence a fluorescence microscope (a light microscope equipped with a light source with different wavelengths) must be used to observe the light-emitting property of the colored product. Source: http://www.doksinet There is a plethora of stains and staining techniques. The stain or technique used depends on the organelle or component of interest. The curious and ambitious student may click on the hyperlinks for more information about these alternative stains and for background information on H&E. Interpreting LM Images In order to analyze an image, you must be able to interpret the numerous clues that you see. The presence or absence of these clues, when put together, should point you in the correct direction. The following clues and suggested interpretations are places to start. You should pay attention to the: • Nuclear Profile o leucocytes tend to have distinctive nuclear profiles

– i.e multilobed, single nucleus of a neutrophil o the nucleus in a contracted smooth muscle cell will appear twisty (corkscrew) • Nuclear Location o skeletal muscle nuclei are located peripherally in the plasma membrane o polarity will be apparent in pancreatic exocrine cells and simple columnar cells • Architecture o simple cuboidal cells arranged in a circle are often indicative of a duct or a gland • Relative Size of the Cells o if you are looking at a peripheral blood smear, note the cell size of other cells relative to the erythrocytes o if you are looking at a spinal cord specimen, the largest neurons visible should be motor neurons Source: http://www.doksinet o if the tissue type is unknown relative size may not be as informative You will definitely add to this and build your own clue library as you progress through medical school and life. LM Self-Assessment ELECTRON MICROSCOPY Electron Microscopy makes it possible for the microanatomist to view ultrastructural

components such as nuclei, mitochondria, endoplasmic reticulum, microvilli, microtubules, etc. Unlike LM, tissues are normally stained with heavy metals such as osmium tetroxide, uranium or lead salts. Due to their differing affinity for these heavy metals some structures appear electron-dense (high affinity) or electron-lucent (low affinity). A fine example of this is the heterochromatin (tightly-coiled chromatin, H) and the euchromatin (loosely-coiled chromatin, E) present in a cell’s nucleus. Before we proceed any further, it is important for you to understand that there are two basic EM techniques known as Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), shown below. Source: http://www.doksinet Source: http://www.doksinet You probably noticed, from the pictures above of a Kuppfer cell (specialised macrophage of the liver sinusoids, responsible for the removal of particulate matter from the circulating blood), that an SEM image (on the right) is

threedimensional. As a result, it is (usually) easier to interpret than a twodimensional TEM image (on the left) but TEM reveals more detail Consequently, it is much more informative. Interpretation of TEM Images As stated above, TEM allows you to view ultrastructural components. Due to this increased resolution, understanding the function of the Golgi apparatus, SER, RER, microvilli, and other ultrastructural components is crucial for TEM image analysis. For example, the presence of lots of Golgi should inform you that the cell is either a secretory cell, such as an antibodyproducing cell known as a plasma cell, or a macrophage which contains numerous lysosomes. Function is relatively easy Making the correct connection between structure and organelle is where the difficulty lies. The following guidelines should be helpful: • Pay attention to magnification. o In high magnification images, lipid bilayers have a trilaminar appearance due to the presence of an external phospholipid

layer, an intermembranous space and an internal lipid layer. • Note the relative sizes of different organelles. o The nucleus is usually the largest intracellular component. • Locate the nucleus. o Surrounded by a lipid bilayer with nuclear pores o May contain an outer ring of heterochromatin (electron-dense) and centrally-located euchromatin (electron-lucent) or a random mixture of the two • Locate other organelles that are surrounded by a lipid bilayer such as mitochondria (look for characteristic cristae) and secretory vesicles. • RER may look like beads (ribosomes) on a string (endoplasmic reticulum). • The Golgi apparatus may be a little hard to identify but it is usually located between the nucleus and the endoplasmic reticulum. Source: http://www.doksinet • Look at the cell’s shape. o Is it misshapen or ragged? If so, it may be a motile cell such as a macrophage or another white blood cell. o Is it somewhat square and in close proximity to similar cells? If so,

it may be an epithelial cell. EM Self-Assessment Practice alone will lead to proficiency. There are many LM and EM image banks on the web but your Wheater’s Functional Histology text should come with a CD that has plenty of images. Use the slide-vision function to see if you can correctly identify the cell’s origins. If you prefer hands on learning, another option is to 1) remove all the required slides for one particular lab from the histology slide box – i.e muscle 2) without looking at the label, select a slide at random 3) place the slide on to the stage 4) look at the image at low, medium and high power 3) make an educated guess about the tissue origin and the components that you see