Histology and Histological Techniques | Animal Husbandry & Veterinary Science Optional for UPSC PDF Download

Stains

1. Definition of Staining:

   • Staining is a process where an element changes color after being treated with certain substances.
   • The stained element usually becomes transparent, meaning you can see through it.

2. Staining Process:

   • The element is treated with reagents (chemicals) or a series of them.
   • The result is a colored appearance, but individual dye particles are not visible.

3. Staining Characteristics:

   • Stained elements are transparent, allowing light to pass through.

4. Staining Non-living Tissues:

   • Non-living tissues, like those from killed organisms, can also be stained.
   • Substances used for staining are absorbed by these tissues, similar to how wool absorbs dyes.

5. Absorption in Staining:

   • The staining substances are taken up by the elements, just like how wool absorbs dyes.
   • This absorption occurs whether or not additional substances called mordants are used.

Chemical Classification of Stains used in Biological Work

1. Types of Organic Stains:

   • Organic stains are divided into three groups: acidic, basic, and neutral stains.

2. Acidic Stains:

   • Used for staining cytoplasm and proteins.
   • Work well at low pH levels.
   • Common acidic stains include picric acid, acid fuchsin, Congo red, Janus green B, and others.

3. Basic Stains:

   • Used to stain the nucleus, chromosomes, and nucleic acids.
   • Common basic stains include basic fuchsin, crystal violet, methylene blue, and haematoxylin.

4. Neutral Stains:

   • Combine properties of both acidic and basic stains.
   • Used for acidophilic and basophilic tissues.

5. Acidophilic Tissues:

   • Tissues or cells that take up acidic stains.
   • For example, the cytoplasm is acidophilic.

6. Basophilic Organelles:

   • Nucleus, chromosomes, and DNA attract basic stains.
   • They are known as basophilic organelles.

7. Metachromasia:

   • Some basic stains can color certain cell components differently from their original color.
   • This property is called metachromasia.
   • Useful for histochemical and physiochemical tests.
   • Stains like thionine, azure A, and toluidine blue display metachromasia and react with substances like nucleic acids and acidic lipids.

Principies of Staining Tissues

1. Staining Definition:

   • Staining is the process of adding color to cells or tissues in animal or plant bodies using specific dyes.

2. Selection of Dyes:

   • The choice of dye depends on the chemical nature of the material, the pH value of the fixative, and the stain's reactivity to the material.

3. Cytological Stains:

   • Most stains used are solutions of aromatic organic compounds with two important chemical groups: chromophoric and auxochromic.

4. Chromophoric Group:

   • This group provides color to the dye.
     
   • Examples include carboxyl (-COOH) and indamin (-N=) groups.

5. Auxochromic Group:

   • This group enables the dye to attach to the tissue, dissolve, and dissociate in water.
   • Examples include hydroxy (-OH) groups.

Mordants

1. Definition:

   • Some dyes can only stain proteins and cytoplasm in the presence of certain metal compounds known as mordants.

2. Chemical Nature of Mordants:

   • Mordants are usually double salts of potassium, ammonium, or ferric sulfate.

3. Composition of a Mordant:

   • A mordant, chemically, is often a double salt containing elements like potassium, ammonium, or ferric sulfate.

4. Formation of "Lake":

   • The combination of the mordant and the stain is collectively called a "lake."

5. Important Mordant:

   • One crucial mordant is iron alum, specifically ferric ammonium sulfate.
   • Iron alum is commonly used with stains like haematoxylin and carmine.

Progressive Stain and Regressive Stain

1. Progressive Stain:

   • Slowly colors tissue elements in a specific order.
   • For example, Ehrlich's haematoxylin first stains the nucleus and then the cytoplasm.
   • By removing the section from the stain bath at the right moment, you can choose to stain only the nuclei or both nuclei and cytoplasm as needed.

2. Regressive Stain:

   • Begins with over-staining the material.
   • The stain is then washed out until it remains only in the cells or specific components under study.
   • The second stage, called "destaining" or differentiation, is crucial.
   • Ehrlich's haematoxylin is an example of a regressive stain.

3. Direct Staining:

   • Dye directly colors tissues perfectly when used as simple aqueous or alcoholic solutions.
   • Examples include methylene blue and eosin.

4. Indirect Staining:

   • Involves using mordants, like haematoxylin.

5. Specific Stains:

   • Certain tissues or cell components stain only with specific dyes.
   • These stains have little or no effect on other tissues or components.
   • Examples: Weigert's elastin stain turns elastic tissue blue to black, and Scharlach R and Sudan black B stain intracellular fat red and black, respectively.

Differential Staining of Cytofplasmic and'connective Tissue Elements

1. Cytochemical Stains for Cellular Components:

   • Certain cellular components take stains based on their chemical compositions and enzymatic activities.
   • Cytochemical stains help detect the chemical nature of various cell components.

2. Proteins:

   • Naphthol yellow S and mercuric bromophenol blue are used to analyze proteins.
   • Stains reveal proteins containing basic amino acids like lysine, arginine, and histidine.
   • Millon's reagent specifically stains the amino acid tyrosine.

3. RNA and DNA:

   • Feulgen solution and methyl green pyronine stain DNA.
   • Methyl green pyronine colors DNA green and RNA red.
   • Azure B is another stain for RNA.

4. Lipids:

   • Lipid contents in cells are stained by fat-soluble dyes like Sudan Black B.
   • Sudan Black B targets phospholipids and the Golgi complex.
   • Other lipid stains include osmium tetroxide and Sudan red.

5. Carbohydrates:

   • Schiff's reagent stains carbohydrates, including starch, cellulose, hemicellulose in plant cells, and mucoproteins and chitin in animal cells. They appear red.

6. Enzymes:

   • Enzymes concentrated in mitochondria are studied using tetrazolium salt.
   • Tetrazolium salt, when acted upon by mitochondrial dehydrogenase enzymes, changes color.

7. Vitamins:

   • Riboflavin, vitamin A, and thiamine are detected using fluorescent dyes like berberine, acridine-orange, yellow, and coriphosphine.

8. Light Microscopy Stains:

   • Both acidic and basic stains are used for light microscopy to stain cells or tissues.
   • Acidic stains highlight cytoplasmic proteins and carbohydrates, while basic stains emphasize the nucleus, chromosomes, etc.

Methods of Processing, and Preparation of Tissues

1. Preparation of Tissues for Microscopic Examination:

   • Before examining body tissues with a microscope, they undergo a series of preparations.

2. Steps in Tissue Preparation:

   • Fixing, hardening, dehydration, de-alcoholization, clearing, embedding, section cutting, mounting, staining, etc.

3. Fixation:

   • Preserves cell structure in a lifelike condition.
   • Functions include setting tissues in a normal position, studying intra-cellular bodies, preventing post-mortem changes, revealing differences in organ parts, and making cell constituents insoluble for further processing.

4. Prompt Fixation Importance:

   • Tissues should be fixed promptly after death or injury to prevent distortion and dehydration.
   • Delay may lead to confusing changes, and keeping tissues in normal saline allows unwanted processes.

5. Fixation Fluids:

   • Various fixatives are used, including formaldehyde, alcohol, acetone, mercuric chloride, chromates, osmic acid, and picric acid.
   • Commonly used is a 10% solution of formaldehyde.

6. Fixation Duration and Temperature:

   • The exposure time to the fixative depends on the temperature.
   • Longer exposure at lower temperatures (0-5°C) is preferred to avoid over-fixation.

7. Rapid Fixation Technique:

   • For quick fixation, a small tissue piece is placed in a 10% formaldehyde solution and warmed.
   • Frozen method: Tissue is fixed within half an hour at about 45°C.
   • Alternatively, tissues can be moved through alcohol and xylene to be ready for embedding in paraffin in an hour.

Paraffin Wax Embedding and Sectioning

(a) Debydration

1. Dehydration Technique:

   • Dehydration is a process in tissue preparation to remove water.

2. Standard Steps:

   • Tissues are fixed and washed by standard methods.
   • They are then immersed in a series of alcohol solutions to dehydrate them gradually.

3. Step-by-Step Dehydration:

   • 60% alcohol for 12-24 hours.
   • 70% alcohol for the same duration.
   • 90% alcohol for the same duration.
   • 96% alcohol for the same duration.
   • Absolute alcohol for the same duration.

4. Use of Specimen Tubes:

   • Preferably, use corked specimen tubes for the dehydration process.
   • Occasional shaking of the tube accelerates alcohol penetration.

5. Duration for Dehydration:

   • Small tissue pieces usually need 12 hours for each change of alcohol.
   • Larger tissues may require 18-24 hours.
   • Hard tissues can be softened using Lendrum's technique, involving a 4% aqueous phenol immersion for 1 to 3 days after washing out fixatives.

6. Rapid Dehydration for Thin Tissues:

   • For thin tissue slices (not more than 5 mm thick), immerse in each 50%, 70%, 90%, and 96% alcohol for half an hour each.

(b) Clearing

1. Clearing Process:

   • In tissue preparation, clearing is a step to replace alcohol with substances like xylol, cedarwood oil, benzene, toluene, or chloroform.

2. Common Clearing Agents:

   • Xylol is the fastest but makes tissues brittle.
   • Cedarwood oil is slow and doesn't harden tissues.
   • Benzene is excellent for delicate tissues, causing minimal shrinkage.
   • Toluene is satisfactory, allowing tissues to be exposed for up to 24 hours without shrinkage.
   • Chloroform has less hardening effect than xylol but takes longer to penetrate tissues.

3. Choosing Clearing Agents:

   • Xylol should be used briefly due to tissue brittleness.
   • Cedarwood oil is slower and doesn't harden tissues, suitable for certain applications.
   • Benzene is the best, causing minimal shrinkage and evaporating from tissues in the paraffin-embedding bath.
   • Toluene is also good for longer exposure without risk of shrinkage.
   • Chloroform is suitable for larger pieces of pathological tissues.

4. Clearing Techniques:

   • Small pieces (up to 5 mm thick) need 15 to 30 minutes in two changes of clearing agents.
   • Larger pieces (up to 1 cm thick) require 1.5 to 3 hours in each of two changes.
   • Bulky specimens, like whole embryos, may need up to six hours in each of two changes.
   • If specimens aren't transparent after the prescribed time, they should stay in the clearing agent until they reach transparency.

(c) Embedding Technique
In the embedding process, transfer the object from the clearing agent to a mixture of paraffin wax and the clearing agent.
Place the tube in the oven set at 50 to 60°C for half an hour to 16 hours, depending on the object's size and nature.
Duration: Half an hour for objects up to 5 mm thick, one hour for 5 mm, two hours for 10 mm, and 8-16 hours for bulky specimens like whole embryos.

1. Further Embedding Steps:

   • Transfer the object to pure paraffin wax in the oven for a quarter to eight hours.
   • Move to another bath of pure paraffin wax for the same duration.

2. Additional Notes:

   • Specimens up to 3 mm thick usually need half an hour in each of the two baths of pure paraffin wax.
   • Specimens 5 mm thick require about an hour, and 1 cm thick requires about 4 hours.
   • Very bulky objects need approximately 8 hours in each of the two wax baths.

3. Considerations for Specific Materials:

   • Pathological materials with blood clots, emboli, striated and non-striated muscles, organs with large blood content, and fibrous tissues should be immersed for the minimum time needed for thorough wax penetration.
   • Prolonged exposure to heat can lead to hardening and shrinkage in these materials.

Casting the Paraffin Blocks

1. Preparing the Mold:

   • Thinly coat the inside of the embedding angles and plates with liquid paraffin.
   • Adhere the angles to the plate to create a mold of suitable size.

2. Filling the Mold:

   • Fill the mold with melted paraffin wax.
   • Place the object in the wax, ensuring it sets in the right position for sectioning.

3. Cooling Process:

   • Once the wax block is partially set, gently immerse it, still in the mold, in cold water.
   • This rapid cooling helps prevent wax crystallization, avoiding crumbling when the block is mounted on the microtome for sectioning.

4. Section Cutting:

   • Learning section cutting is best acquired through practical experience under skilled guidance in a laboratory.
   • Detailed guidance on section cutting is not provided here.

Mounting Sections on slides and Hydrating

1. Preparing the Slide:

   • Dampen the fingertip with glycerin albumen (Mayer).
   • Create a thin smear on the slide, large enough for the section.

2. Placing the Section:

   • Use a needle or forceps to pick up the section and place it on the albumen.

3. Flattening the Section:

   • Gently press the section onto the slide with the thumb.
   • Ensure it lies flat without folds or creases, avoiding any damage to the section.
   • If sections are curled or folded, apply a drop of 1% potassium dichromate, then gently heat until the section floats flat. Blot the edges to remove excess solution.

4. Removing Wax:

   • Gently warm the slide until the paraffin wax just melts.
   • Wash away wax with two or three changes of alcohol.

5. Xylene and Alcohol Wash:

   • Remove xylene by thoroughly washing with absolute alcohol.
   • Wash with two changes of 90% alcohol.
   • Wash with 70% alcohol.

6. Final Wash:

   • Wash with two changes of distilled water for aqueous stains.
   • For alcoholic stains, staining can begin immediately after washing with 70% alcohol.

7. Staining Process:

   • Proceed with staining based on the desired technique.

8. Note for Opalescent Appearance:

   • If the section appears opalescent (indicating water presence), backtrack each step until the preparation is no longer opalescent when brought down to alcohol.

Preparation and Staining of Blood Films

1. Blood Collection:

   • Collect a drop of blood from the jugular or ear vein onto a thick microscopic glass slide.
   • Spread the blood drop into a thin film using another slide's edge.
   • Ensure the smear edges are within the glass surface.
   • Allow the smear to air-dry, or fix it in methyl alcohol before staining.

2. Stains for Blood Cells:

   • Various stains are used for blood cell studies, important for phagocytosis and studying bacteria causing septic conditions.
   • Common stains include Romanowsky and Giemsa, available in tablet or powder form.

3. Wright's Stain:

   • Wright's stain is reliable for routine work.
   • It can be purchased in liquid or powder form.
   • To use, mix with a proper solvent to create a stock solution.
   • Apply the solution to a blood smear, let it stand for 1 minute.
   • Add distilled water until a metallic lustre appears.
   • Allow the stain to act for 5 minutes, then wash off, dry, and examine.
     Note: Carefully wash to remove the metallic sheen precipitate.

4. Giemsa's Stain:

   • Apply a fixing agent to moist films for 12 hours (95% ethyl alcohol and saturated aqueous HgCl₂).
   • Wash with water for a few seconds.
   • Apply Lugol's solution for 6 minutes.
   • Wash in water and then in 0.6% sodium thiosulfate.
   • Stain with Giemsa for 8 to 10 hours.
   • Wash and mount for examination.

Celloidin Embedding

Celloidin embedding is a method used for preserving the relationships of different cell layers in tissues, especially in the eye, for large objects, pieces of the central nervous system, and hard tissues like decalcified bone. There are two methods: the wet method and the dry method.

Wet Method:

• Suitable for preserving tissues with varying consistencies.
• Effective for maintaining the structure of different cell layers in the eye.
• Ideal for larger objects and pieces of the central nervous system.
• Particularly useful for hard tissues like decalcified bone.

Dry Method:

• Also employed for embedding tissues in celloidin.
• Suitable for preserving tissues with different consistencies.
• Commonly used for hard tissues like decalcified bone.

Celloidin Paraffin Wax (Double Embedding):

• Used for embedding tissues in celloidin and paraffin wax for serial sections.
• Helpful for studying tissues in a sequential manner.

Celloidin Pyridine (Rapid Method):

• A quick method for dehydrating, clearing, and embedding tissues.
• Eliminates the need for alcohols, preventing tissue hardening.
• Especially useful for preserving tissue structure efficiently.

By using celloidin embedding, scientists can maintain the natural relationships of cells in various tissues, making it a valuable technique in the study of complex structures like the eye and large organs. The choice between wet and dry methods depends on the specific characteristics of the tissues being studied.

Freezing Microtomy

Freezing microtomy is a method used for specific purposes such as identifying fat in tissues, certain impregnation methods for the central nervous system, and quickly examining pathological material like tumor pieces. This technique allows for the cutting of thin frozen sections, offering an advantage of less shrinkage compared to paraffin-embedded material.

Key Points:

1. Purposeful Usage:

   • Employed for specific purposes like identifying fat, certain central nervous system studies, and rapid examination of pathological material.
   • Not a substitute for paraffin embedding.

2. Advantage of Reduced Shrinkage:

   • Provides thinner sections with lesser shrinkage compared to paraffin-embedded material.

3. Limitations:

   • Cannot be used for cutting serial sections.
   • Sections cannot be stored as with paraffin-embedded material.

4. Manipulation Similar to Celloidin:

   • Handled similarly to celloidin sections, but extra care is needed due to the absence of an embedding mass.

5. Special Microtome:

   • Requires a special microtome for cutting frozen sections.

6. Procedure:

   • Tissues for histochemical or cytochemical observations are embedded in ice and sectioned using a freezing microtome.
   • The tissue is placed on a stage, water drops are sprinkled on it, and carbon dioxide is used to freeze the tissue.
   • The frozen tissue is then sectioned using steel razors or razor blades.

7. Improved Freezing Microtome:

   • Some microtomes are enclosed in a low-temperature chamber, with tissues cooled by liquid carbon dioxide.

8. Versatility:

   • Suitable for cutting sections of both fixed and fresh tissues.

By using freezing microtomy, scientists can quickly examine tissues, especially in surgical settings, but it's crucial to note its specific applications and limitations compared to other embedding methods like paraffin embedding.

Microscopy 

1. Cell Size Challenge:

   • Cells in animals, plants, and bacteria are too small to be seen with the naked eye.
   • The diameter of most cells ranges from 1.0 micrometer (μm) to 1.0 millimeter (1.0 mm = 1,000 μm).

2. Need for Instruments:

   • Artificial aids or observational instruments are necessary to magnify and focus cells because the human eye can't distinguish objects closer than about 0.1 mm or 10 micrometers.

3. Challenges with Living Cells:

   • Living cells are transparent, making it challenging to observe their components.
   • Early cytologists used various methods like slide preparation, involving cutting tissues into thin slices, dehydrating, staining, and mounting to enhance visibility.

4. Staining for Contrast:

   • Staining cells helped create contrast among different components, making them more distinguishable.

5. Role of Lenses:

   • Magnification of cells and their components was achieved using lenses.
   • Lenses, although limited in magnification, were combined to form microscopes.

6. Microscope Definition:

   • The microscope, derived from Greek ('mikros' meaning small and 'skopein' meaning to see), is an instrument designed to magnify and observe minute objects.

In essence, microscopes are essential tools in biology, allowing scientists to study the intricate details of cells and their components, making the invisible visible.

Resolving Power in Microscopes Simplified:

1. Definition of Resolving Power:

   • Resolving power refers to an observational instrument's ability to reveal structural details, expressed as the smallest distance allowing two points to be observed as distinct.

2. Limit of Human Eye:

   • The unaided human eye, even under optimal conditions, cannot distinguish points closer than about 0.1 millimeter apart in green light.

3. Magnification and Resolution:

   • Magnification, the increase in image size, is useless without proper resolution to distinguish structure details.
   • Increased magnification without improved resolution results in a large but blurred image.

4. Human Eye and Magnification:

   • The human eye has limited magnification capability; magnifying glasses can provide up to about 10 times magnification.
   • A light microscope, commonly used, has a useful magnification of about 1,500 times.

5. Abbe's Relationship for Resolution:

   • Abbe's relationship, a formula, calculates the limit of resolution in microscopes.
   • It involves factors like the wavelength of light used, refractive index, and aperture angle.

6. Numerical Aperture (NA):

   • The numerical aperture (NA) is a measure of the lens angle, indicating the amount of light it can gather.
   • Higher NA and shorter wavelength result in lower resolution and higher microscope resolving power.

7. Manipulating Factors for High Resolution:

   • Achieving high resolution involves manipulating three factors: wavelength of light, refractive index, and aperture.
   • The aperture angle is limited, usually less than 90°, due to potential lens distortions.
   • Immersion oils with higher refractive indices enhance resolution, but there are limits to altering the refractive index.

In essence, achieving high resolution in a microscope requires understanding and optimizing these factors to reveal finer details in microscopic structures.

Improving Microscope Resolution Simplified:

1. Wave-Length and Resolution:

   • Microscopes can't resolve objects smaller than half the wavelength of light. Average white light has a wavelength of about 0.55 μm.
   • Microscopes using white light can't resolve objects less than 2500 Å (0.25 μm).

2. Enhancing Resolution:

   • Resolution improves by using shorter wavelength light and achromatic lenses.
   • For instance, using violet light with a wavelength of 4000 Å gives a resolution limit of 1700 Å (0.17 μm).
   • Infrared radiation with a wavelength of 8000 Å provides a resolution limit of 0.4 μm.

3. Media for Improved Microscopes:

   • Traditional glass lenses are no longer transparent with shorter wavelengths.
   • Improved microscopes use refractive media like quartz lenses and reflecting optical instruments.

4. Microscope Types:

   • Historically, microscopes used sunlight for illumination.
   • Over time, advancements have led to various microscope types with different light sources.

In summary, using shorter-wavelength light and advanced lenses enhances microscope resolution, allowing scientists to study smaller structures with greater clarity. Various microscope types have evolved over the years, incorporating improved technologies for better observations.

(a) Bright Field Microscope
Compound Microscope Simplified:
Fig. Modern Compound microscope

1. Overview of Compound Microscope:

   • The compound microscope of the 20th century is a greatly improved and modified version.
   • It incorporates three lens systems for better functionality.

2. Lens Systems: 

   • Condenser Lens System:
     Located beneath the specimen on the microscope's stage.
     Gathers and focuses light rays onto the specimen.

   • Objective Lens System:
     Positioned near and above the specimen.
     Creates and magnifies an image of the specimen.

   • Eyepiece Lens System (Ocular Lens):
     Located close to the observer's eyes.
     Magnifies and forms a secondary image of the primary image produced by the objective.

In simple terms, the compound microscope uses a combination of lenses to collect, focus, and magnify light, enabling detailed observation of specimens placed on the stage. The three lens systems work together to provide a clearer and more magnified view of microscopic objects.

(b) Electron Microscope:

1. Limitation of Light Microscope:

   • Light microscopes have limitations and can only resolve objects close to the wavelength of light used.
   • Objects between 2,000 Å and 3,000 Å in diameter may not be sharply resolved.

2. Search for Improved Source of Light:

   • Biologists sought a light source with a shorter wavelength.
   • Electrons were identified as a potential source, produced by heating a metal filament in a vacuum tube.

3. Electron Beam Properties:

   • Electrons, like light, have both particle and wave characteristics.
   • Electron beams have a very short wavelength (0.05Å) compared to light rays (5500Å).

4. Electron Microscope Invention:

   • Electron microscopes were independently designed by Kuoll and Ruska (Germany, 1932), Marton (Belgium, 1934), and Prebus and Milller (Canada, 1934).

5. Working Principle:

   • Electron microscopes work on the principle of focusing and controlling accelerated electrons using electromagnetic lenses.
   • To avoid electron collisions, a high vacuum condition is crucial.

6. Types of Electron Microscopes:

   • Early electron microscopes that utilize transmitted electrons are known as transmission microscopes.

In essence, electron microscopes, born out of the need for higher resolution, use accelerated electrons in a vacuum to achieve magnification and detailed imaging of specimens, surpassing the limitations of light microscopes.

Transmission Electron Microscope (TEM):

1. Working Principle:

   • In the transmission electron microscope (TEM), a cathode emits an electron beam in a highly evacuated cathode tube.
   • Electrons are collected and focused on the object using an electromagnetic condenser lens.
   • After passing through the object, electrons are collected by an electromagnetic coil acting as an objective lens.
   • A third electromagnetic lens acts as an ocular or projection lens to magnify the image.

2. Image Visualization:

   • The final magnified image can be viewed on a fluorescent screen or recorded on a photographic plate.
   • Scattering of electrons from molecular constituents of the cell produces the image, with greater scattering in denser materials.

3. Resolving Power:

   • Anatomical-type electron microscopes have a resolving power 400 times (theoretically 2000) that of a light microscope.
   • Used for studying ultrastructures of cells and their components, contributing to a deep understanding of cell biology.

Scanning Electron Microscope (SEM):

1. Principle:

   • SEM is a recent development where a fine electron beam scans the specimen's surface, similar to a television screen scanning.
   • Secondary electrons are emitted from the surface where the beam scans and collected by a positively charged grid.

2. Image Formation:

   • The signal from the grid is transferred to a television tube, synchronized with the beam in the microscope.
   • An image of the specimen is produced on the screen, resembling how our eyes collect light reflected from solid objects.

3. 3D Appearance:

   • SEM images have a remarkable three-dimensional appearance, enhancing our understanding of the surface structures of specimens.

Fig. Conaparison of optical pathways in light and electron mieroscopes

In the last we can summarize the uses of difforent types of microscopes in diflerent fields of biology in the following Table.

Table: Uaes dfdifterent mieroscopos for different etructurcs of llving body

Method of Measurement of Cells

Light Microscope:

1. Cell Size and Units:

   • Cells are microscopic, measured in micrometers (um), microns (u), and Angstroms (Å).
   • Light microscopes are commonly used for cell measurements.

2. Measurement Method:

   • Ocular micrometer disc and stage micrometer are used in light microscopes.
   • Ocular disc is a glass plate on the ocular diaphragm calibrated for each lens and objective.
   • Stage micrometer, a marked glass slide, helps calibrate the ocular disc by setting the zero line and reading known intervals.

3. Calibration Process:

   • The units of both ocular disc and stage micrometer are matched.
   • Calibration involves setting zero on the stage micrometer, aligning it with the disc's zero, and reading across the scale.
   • The size of viewed objects is then determined by dividing the dimension on the ocular disc by the magnification factor.

Electron Microscope:

1. Importance of Magnification:

   • Magnification is crucial in interpreting cell structure in electron micrographs.
   • Grating replica, with known lines per inch, is used to determine electron microscope magnification.

2. Grating Replication:

   • An exposure is made on a photographic plate using the grating replica in the electron microscope.
   • The plate is developed, and a straight edge marker measures the distance between the outer limiting grating lines.

3. Measurement Units:

   • Electron microscope measurements are often reported in Angstrom units due to high magnification.
   • Detailed structures can be measured with a millimeter ruler when the magnification is known.
   • Cell structure size in Angstroms = (Size in mm) x 10^7 / Magnification.