1-Nature of Light (Wave-Particle Duality):

Light has a dual nature, meaning it behaves both as a wave and as a particle
depending on how it is observed. This concept is essential to understanding how
light interacts with matter.
Wave Nature of Light
Light acts as a transverse electromagnetic wave, with electric and magnetic fields
oscillating perpendicular to each other. It shows wave-like properties such as:
 Reflection and Refraction
 Diffraction and Interference
 Dispersion (like a rainbow from a prism)
 Polarization
✅ Example: Light passing through a prism splits into colors due to different
wavelengths bending at different angles.
Particle Nature of Light
Light also consists of particles called photons, each carrying a specific amount of
energy depending on its frequency. This explains:
 The photoelectric effect (ejection of electrons from metal)
 How energy is transferred in small, discrete packets
E.g: When ultraviolet light shines on a metal, it can eject electrons, showing light's
particle behaviour.
2--Main Components of a Microscope[L/Q]
A microscope has several key components, divided into two main categories: optical
parts (involved in image formation) and mechanical parts (used for support and
adjustment).
🔬 Optical Components:
1. Eyepiece (Ocular lens):
The lens you look through, usually with 10x magnification.
2. Objective lenses:
Located on the rotating nosepiece; usually 4x, 10x, 40x, and 100x (oil
immersion).
3. Condenser:
Focuses light onto the specimen.
4. Iris diaphragm:
Controls the amount of light passing through the specimen.
5. Light source (Illuminator):
Provides light to view the specimen; can be a mirror or built-in LED/bulb.
⚙️Mechanical Components:
1. Base:
The bottom support structure of the microscope.
2. Arm:
Connects the base to the head and is used to carry the microscope.
3. Stage:
The platform where the slide is placed. May include stage clips or a
mechanical stage to hold and move the slide.
4. Coarse adjustment knob:
Moves the stage up and down for general focusing (used with low power
lenses).
5. Fine adjustment knob:
Makes small focusing adjustments (used with high power and oil lenses).
6. Nosepiece (Turret):
Rotating part that holds and switches between objective lenses.
7. Body tube (Head):
Connects the eyepiece to the objective lenses.
3-How an Image is Formed in a Microscope
In a compound microscope, image formation occurs through two main lenses: the
objective lens and the eyepiece (ocular lens).
1. Light from the illuminator passes through the specimen and is focused by the
condenser.
2. The objective lens captures this light and forms a real, inverted, and
magnified image of the specimen inside the body tube.
3. The eyepiece lens then magnifies this real image to produce the final image.
4. The final image seen by the observer is virtual, highly magnified, and
inverted.
5. Total magnification is calculated by multiplying the magnification of the
objective and eyepiece lenses.
Example: 10x eyepiece × 40x objective = 400x magnification.
4-What is Histotechnology?
Histotechnology is the science of preparing tissue samples for microscopic
examination to study the structure and function of cells and tissues. It plays a vital
role in diagnosing diseases, especially cancers and infections.
🔬 Key Tasks in Histotechnology:
1. Fixation: Preserving tissue to prevent decay.
 2. Processing: Dehydrating and embedding tissue in paraffin wax.
3. Sectioning: Cutting thin tissue slices using a microtome.
4. Staining: Coloring tissue with dyes (like H&E stain) to highlight different
structures.
5. Microscopy: Examining stained slides under a microscope.
5: Types of Microscopic Lenses
Microscopes use different types of lenses to magnify and focus the image of a
specimen. The main types of microscopic lenses are:
1. Eyepiece Lens (Ocular Lens):
The lens you look through, usually with 10x magnification.
2. Objective Lenses:
Located on the rotating nosepiece; common magnifications are 4x, 10x, 40x,
and 100x (oil immersion). These are the main magnifying lenses.
3. Condenser Lens:
Focuses light onto the specimen for clearer viewing, located below the stage.
4. Field Lens:
Part of the eyepiece system that helps to focus and direct light through the
eyepiece.
5. Tube Lens (in advanced microscopes):
Found in some modern optical systems, it focuses light from the objective to
the eyepiece.
6--Advantages of Lenses in a Microscope
1. Magnification:
Lenses enlarge small specimens, making microscopic details visible to the
human eye.
2. Clarity and Resolution:
High-quality lenses improve image sharpness and allow clear differentiation of
fine structures.
3. Accurate Diagnosis:
Lenses help in examining cells and tissues for medical and scientific
purposes, aiding accurate diagnoses.
4. Adjustable Viewing:
Using different objective lenses (e.g., 4x, 10x, 40x, 100x), users can switch
magnifications as needed.
5. Color Correction:
Advanced lenses like achromatic and apochromatic lenses reduce color
distortion for clearer images.
 7
Feature Achromatic Lens Apochromatic Lens
Chromatic Aberration Corrects 2 wavelengths Corrects 3 wavelengths
Correction (typically red and blue) (red, green, and blue)

Spherical Aberration Partial correction Better correction,

Correction especially at higher
magnification
Image Quality Good image quality Superior image quality
with better color accuracy

Cost Less expensive More expensive

Use Common in basic and Used in research and
mid-range microscopes high-precision microscopy

Label on Objectives Usually labelled as Labelled as "Apo" or

"Achromat" "Apochromat"

8-Merits and demerits of an achromatic lens

Merits:
 Reduces chromatic aberration for two wavelengths (red and blue)
 Provides clearer images than simple lenses
 More affordable than apochromatic lenses
 Widely used in basic optical instruments
 Simple design and easy to manufacture
 Suitable for general laboratory and field use
Demerits:
 Does not fully correct chromatic aberration (green may remain uncorrected)
 Lower image resolution compared to apochromatic lenses
 May still exhibit spherical aberration at image edges
 Not suitable for high-precision or research-level imaging
 Limited colour fidelity in high-magnification work

9--How different samples are handled in fixation based on thickness

Tissue
Examples Handling Strategy Fixation Time
Thickness

1–3 mm Biopsies (skin, Fix immediately to prevent

6–8 hours
(Thin) mucosa) drying. Use small containers.

Cut uniformly. Avoid

4–6 mm Liver, kidney, breast
folding/stacking. Use enough 12–24 hours
(Medium) slices
fixative.

Whole organs 24–48 hours

>6 mm Slice into ≤1 cm sections. Brain
(brain, uterus, (brain: 1–2
(Thick) fixed whole, then sliced.
tumors) weeks)

10--Steps of Tissue Processing

Tissue processing involves three main steps, typically done in an automatic tissue
processor over 6–12 hours (or overnight).
1. Dehydration
 Purpose: Removes all water from the tissue.
 Why? Paraffin is not water-soluble.
 How? Tissue is passed through increasing concentrations of alcohol:
o 70% → 80% → 90% → 100% ethanol (absolute alcohol)
 Each step gradually replaces water with alcohol.
2. Clearing
 Purpose: Removes alcohol and prepares tissue for wax infiltration.
 Why? Alcohol is not miscible with paraffin wax.
 How? Use clearing agents that are miscible with both alcohol and paraffin:
o Common clearing agent: Xylene
 The tissue becomes translucent (cleared).
3. Infiltration
 Purpose: Saturates the tissue with molten paraffin wax.
 Why? Wax provides rigidity for section cutting.
 How? Tissue is placed in molten paraffin wax at 58–60°C for several hours.
 Wax replaces the clearing agent in all tissue spaces.
✅ Types of Tissue Processing in Histopathology:
 1. Manual Tissue Processing
– All steps (dehydration, clearing, infiltration) are done by hand using
separate containers.
– Time-consuming and operator-dependent.
2. Automatic Tissue Processing
– Uses a tissue processor machine to perform dehydration, clearing, and
wax infiltration automatically.
– Can be overnight (12–16 hours) or rapid (1–3 hours).
3. Microwave Tissue Processing
– Uses microwave energy to speed up processing steps.
– Much faster (completed in 30–90 minutes).
– Requires special equipment.
4. Vacuum Tissue Processing
– Uses pressure and vacuum cycles to enhance infiltration, especially for
dense or fatty tissues.
– Often used in combination with automatic processors.
5. Rapid (Express) Processing
– Used for urgent biopsies (e.g., cancer diagnosis).
– Uses high temperatures, agitation, or vacuum to reduce time.

11-Purpose of Clearing:
 To remove alcohol (from dehydration).
 To make the tissue transparent (cleared).
 To prepare tissue for infiltration with paraffin wax, which is not miscible with
alcohol.
Why is it called "clearing"?
 The clearing agent refracts light similarly to proteins, making tissues look
transparent or “cleared”.
🧪 Properties of a Good Clearing Agent:
Property Importance

Miscible with alcohol and paraffin To ensure smooth transition

Non-reactive with tissue To avoid damage

Rapid penetration For faster processing

Volatile So it can be easily removed in later steps

Names of common clearing agents:
 Xylene
  Toluene
 Chloroform
 Benzene
 Cedarwood oil
 Clove oil
 Limonene-based agents (e.g., Histo-Clear®)

🎨 Classification of Stains in Histopathology

Stains are classified based on chemical nature, target, and purpose. Here’s a
detailed classification:

🧪 I. Based on Chemical Nature

Type Description Examples

Stain basic (alkaline) components

Acidic stains Eosin, Orange G
like cytoplasm

Stain acidic components like

Basic stains Hematoxylin, Methylene blue
nucleus (DNA/RNA)

Romanowsky stains
Neutral stains Combo of acidic + basic stains
(Leishman, Giemsa)

Amphoteric Hematein (oxidized

Can act as either acidic or basic
stains hematoxylin)

🎯 II. Based on Target Structure

Type Targets Examples

Nuclear stains Nucleus Hematoxylin, Feulgen

Cytoplasmic stains Cytoplasm Eosin, Orange G

Connective tissue Collagen, muscle,

Masson’s Trichrome, Van Gieson
stains reticulin

PAS (Periodic Acid-Schiff), Alcian

Carbohydrate stains Glycogen, mucins
blue

Sudan III, Sudan Black B, Oil Red

Lipid stains Fat droplets
O
Type Targets Examples

Microorganism stains Bacteria, fungi Gram stain, Ziehl-Neelsen, GMS

🎯 III. Based on Purpose

Type Use Examples

Routine stains Basic tissue structure H&E (Hematoxylin & Eosin)

Special stains Specific components PAS, Masson’s Trichrome

Detect chemical
Histochemical stains PAS, Perls’ Prussian blue
substances

Used in cytology for cell PAP stain, Romanowsky

Cytochemical stains
components stains

Immunohistochemical Use antibodies to detect DAB staining,

stains antigens Immunofluorescence