HISTOLOGY:Made Absolutely

Easy

Objectives:How to Use this Presentation:

• It serves as a great review – especially if you havealready read your text

• It is a great way to revise for your exam.• As an adjunct to your regular classes.• To clear any doubts that you may have• I 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 Histology

• The Cell

• Epithelial Tissue

• Connective Tissue

• Cartilage and Bone

• Blood

• Muscle Tissue

• Nervous Tissue

Table of Contents Contd.(b) The Organs:

• Circulatory System• Lymphoid System• Integumantary System• Digestive System• Respiratory System• Urinary System• Endocrine System• Male reproductive System• Female Reproductive System• Organs 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.

Fixation

Dehydration

Embedding

Sectioning

Mounting Staining Observation

(Junqueira’s Basic Histology)

(1) Fixation:

• Purpose: Maintenance of tissue architecture by cross linking proteins and inhibiting autolysis.

Procedure: A large specimen is received

It is cut to smaller pieces

For 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 concentrationsof alcohol beginning with 50% to 100%. Each step takes about2 to 3 hours.

• Clearing: This involves removing the alcohol and replacing itwith a chemical that is miscible in both alcohol and paraffin.The chemical is Xylene solution which will now infiltrate thetissues. Smaller tissues take upto an hour. Larger ones require2 to 4 hours.

(3) Embedding:

• Tissues are then placed in an oven containing liquid Paraffin that “Infiltrates” it.

• The high temperatures of 52-60⁰C 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 with

• Disadvantages: 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 5-10μm thick

• E/M: 0.02-0.1μm 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 hematoxylin

Remaining cytoplasm stains with Eosin

Nuclei are stained with hematoxylin

Muscle fibers stained with Eosin

Courtesy: Color Textbook of histology

Microscope: Types of Light Microscopes:1. Bright-field microscopy: Widely used

by histology students. Involves use offixed and then stained slides to viewslides under an ordinary light.

2. Phase contrast microscopy: usesmodified objective lenses andcondenser to allow the viewing ofliving tissue without prior fixing orstaining.

3. Fluorescence microscopy: used toview inherently fluorescent substancesor those tissues that have beenlabeled by fluorescent stains. Uses alight of a different wavelength (UVlight) is focused onto the cells which inturn emit light in the visible spectrumthat can be viewed.

4. Confocal microscopy: a bright-fieldmicroscope uses large light source toproject light thus reducing the contrastin the image and hence poorresolution. The confocal microscopetherefore uses a small yet intensesource of light- the laser and also aplate bearing a pin-hole aperture toreduce scattering of light

Light microscope has a maximum resolving power of approximately 0.2 μm. 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 Procedures• Autoradiography: 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. Enzymesthat can be viewed like this include: Phosphatase, dehydrogenase and peroxidase.

• Immunocytochemistry: uses fluoresceinated antibodies and anti-antibodies toprovide 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 Microscopewhich uses thin sections stained with heavy metal salts and a beam of electrons isdirected towards it. (b)SEM: Scanning Electron Microscope: which uses wholesections that are stained with a thin layer of gold and then studied with a beam ofelectrons to obtain a three dimensional image.

Courtesy: Color Textbook of histology

Interpretation of the Slides:

Key:A: Longitudinal sectionB: Tangential sectionC: Oblique sectionD, E & F: Transverse sections

Key:A& B: Longitudinal sectionsC: Tangential sectionD & E: Transverse sectionF: Tangential sections

diFIORE’S ATLAS OF HISTOLOGY WITH FUNCTIONAL CORRELATIONS

diFIORE’S ATLAS OF HISTOLOGY WITH FUNCTIONAL CORRELATIONS

Artifacts 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: