histology made easy: chap 1: introduction to histology

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HISTOLOGY: Made Absolutely Easy

Objectives:How to Use this Presentation:

It serves as a great review especially if you have already read your textIt is a great way to revise for your exam.As an adjunct to your regular classes.To clear any doubts that you may haveI have basically tried to consolidate a lot of the data about histology on the web in one place

Good Luck and Enjoy!!!

Table of Contents:(a) The Tissues:Introduction to HistologyThe CellEpithelial TissueConnective TissueCartilage and BoneBloodMuscle TissueNervous Tissue

Table of Contents Contd.(b) The Organs:Circulatory SystemLymphoid SystemIntegumantary SystemDigestive SystemRespiratory SystemUrinary SystemEndocrine SystemMale reproductive SystemFemale Reproductive SystemOrgans of special senses.

Introduction to Histology:Definition of Histology:

Histo comes from Greek which means web or tissue

Introduction To Microscopes:Histology requires the use of Microscopes to view the structures under increasing magnifications. This requires preparation of slides that will then be viewed under microscopes.

How to Prepare a SlideIn order to view the cellular components of a tissue, it has to be processed in a step wise manner to produce a slide which can be viewed under a microscope.FixationDehydrationEmbeddingSectioningMountingStainingObservation

(Junqueiras Basic Histology)(1) Fixation:Purpose: Maintenance of tissue architecture by cross linking proteins and inhibiting autolysis. Procedure:

A large specimen is receivedIt is cut to smaller piecesFor L/M it is dipped in formalinE/M: is dipped in Glutraldehyde and then Osmium tetroxide.Takes 24 hours(2) Dehydration & Clearing:Purpose: To remove all the water because the Paraffin (embedding medium) is immiscible in water.

Dehydration: Solution is placed in increasing concentrations of alcohol beginning with 50% to 100%. Each step takes about 2 to 3 hours.

Clearing: This involves removing the alcohol and replacing it with a chemical that is miscible in both alcohol and paraffin. The chemical is Xylene solution which will now infiltrate the tissues. Smaller tissues take upto an hour. Larger ones require 2 to 4 hours.(3) Embedding:Tissues are then placed in an oven containing liquid Paraffin that Infiltrates it.The high temperatures of 52-60C evaporates the Xylene.A block of Paraffin obtained.L/M: uses Paraffin and plastic resins.E/M : uses resins as embedding medium.

Advantages of Paraffin: it stains reliably and is easy to work withDisadvantages: slices cannot be cut very thinly.

(4) Sectioning:Trimming is the process in which excess paraffin is removed from the block to expose the tissue to be cut by the microtome.Sectioning involves using a machine called a microtome that cuts sections very thinly in the form of ribbons .Sections are cut from these ribbons and mounted on to the glass slide.L/M: the sections are usually 5-10m thickE/M: 0.02-0.1m thick.

The disadvantage of thick sections: Overlapping and therefore decreased resolution.

(5) Mounting & Staining:Mounting: the thin sections obtained from the microtome are mounted upon glass slides.Staining: The most common stains are Hematoxylin and Eosin.

Hematoxylin: Is a BASIC dye.It colors the Acidic Components of cells giving them a bluish tint.Egs. Of structures stained by it are usually protein rich areas like Nuclei, RER. And also Extracellular matrix like the collagen matrix.

Eosin: Is an Acidic dye.It colors the Basic Components of cells giving them a pinkish tint.Egs. cytoplasm, Collagen fibres, Mitochondria, lysosomes, muscle, connective tissue, colloid, red blood cells.

Nuclei are stained with hematoxylinRemaining cytoplasm stains with EosinNuclei are stained with hematoxylinMuscle fibers stained with Eosin

Courtesy: Color Textbook of histologyMicroscope:

Types of Light Microscopes:Bright-field microscopy: Widely used by histology students. Involves use of fixed and then stained slides to view slides under an ordinary light.Phase contrast microscopy: uses modified objective lenses and condenser to allow the viewing of living tissue without prior fixing or staining.Fluorescence microscopy: used to view inherently fluorescent substances or those tissues that have been labeled by fluorescent stains. Uses a light of a different wavelength (UV light) is focused onto the cells which in turn emit light in the visible spectrum that can be viewed.Confocal microscopy: a bright-field microscope uses large light source to project light thus reducing the contrast in the image and hence poor resolution. The confocal microscope therefore uses a small yet intense source of light- the laser and also a plate bearing a pin-hole aperture to reduce scattering of lightLight microscope has a maximum resolving power of approximately 0.2 m. This power is sufficient to magnify the image 1000-1500 times.Different Parts of the Microscope:

Eyepiece: The lens through which the viewer looks at the specimen. Magnifies image 10X.Body tube (Head): Connects the eyepiece to the objective lenses.Arm: Connects the body tube to the base of the microscope.Nosepiece: A rotating disc that bears objective lenses of varying magnifications.Objective lenses: Used to magnify the specimen. A standard microscope has objective lenses of 4X , 10X, 40X upto 100X. Stage: The flat platform where the slide is placed.Aperture: The hole in the center of the stage that allows light to reach the specimen.Stage clips: Metal clips that hold the slide in place.Iris diaphragm: Adjusts the amount of light that reaches the specimen.Coarse adjustment: moves the stage up and down in greater increments. Fine adjustment: Fine tunes the focus by the moving the stage in smaller increments. Stage Control: Moves the stage left and right.Condenser: Collects and focuses light from the illuminator onto the specimen.Illumination: The light source for a microscope.Base: Supports the microscope and bears the illumination.On/off switch: Switch on the base of the microscope to turn the light source on and off.

How to use a microscope:

1. Turn the light on, adjust the brightness.2. Examine the slide by eye by viewing it through the eyepiece.3. Put the x4 objective in position,4. Put the slide on the stage,5. Focus with the coarse focus knob6. Once specimen is visible, then use the fine focus to clearly view it. 7. Fine-tune the condenser and the iris diaphragm to adjust the amount of light reaching the specimen.8. Examine the specimen.9. Examine the slide with the x10 objective and x40 objective using the steps 3-8 with the respective lenses.10. Once finished replace the slide back in the slide box.11. Turn off your microscope.

Advanced Visualization ProceduresAutoradiography: is a method that uses the incorporation of radioactive isotopes into macromolecules.Histochemical (or cytochemical) technique: is a method for localizing cellular structures using a specific enzymatic activity present in those structures. Enzymes that can be viewed like this include: Phosphatase, dehydrogenase and peroxidase.Immunocytochemistry: uses fluoresceinated antibodies and anti-antibodies to provide more precise intracellular and extracellular localization of macromolecules. It involves direct and indirect immunocytochemistry techniques.Electron Microscopes: are of 2 types: (a) TEM: Transmission electron Microscope which uses thin sections stained with heavy metal salts and a beam of electrons is directed towards it. (b)SEM: Scanning Electron Microscope: which uses whole sections that are stained with a thin layer of gold and then studied with a beam of electrons to obtain a three dimensional image.

Courtesy: Color Textbook of histologyInterpretation of the Slides:

Key:A: Longitudinal sectionB: Tangential sectionC: Oblique sectionD, E & F: Transverse sectionsKey:A& B: Longitudinal sectionsC: Tangential sectionD & E: Transverse sectionF: Tangential sectionsdiFIORES ATLAS OF HISTOLOGY WITH FUNCTIONAL CORRELATIONS

diFIORES ATLAS OF HISTOLOGY WITH FUNCTIONAL CORRELATIONSArtifacts in the Slides:Are imperfections in the technique of slide preparation and must not be thought of as a feature of the tissue.

Wrinkles or folds: well-defined dense-staining regions in the section where detail is obscured.

Foreign body introduction

Irregular distribution of staining

A torn slide:

Thank you:Next Slides will discuss The Cell