Microscopy – Principle and Types ( For 7th Semester)

Microscopy – Principle and Types ( For 7th Semester)

1. Microscopy – Principle and Types Dr Showkat Ahmad Wani

2. What is Microscopy? Microscopy is the science of using microscopes to view objects that cannot be seen with the naked eye. It involves two important concepts: Magnification – How much larger the image appears compared to the actual object. Example: 100× means the image is 100 times larger than the real size. Resolution (resolving power) – The ability to distinguish two closely placed points as separate. A microscope with good resolution can show fine details clearly.

3. Principle of Microscopy

4. Types of Microscopes Let’s now look at the main types one by one — from the simplest to the most advanced.

5. Simple Microscope Principle: Uses a single convex lens to magnify the object, just like a magnifying glass. Construction: Only one lens (simple design). Working: The object is placed close to the lens. The lens bends (refracts) light rays to form an enlarged, virtual image on the same side as the object. Magnification: Up to about 10× to 20× . Example: Magnifying glass, hand lens. Used for: Observing small insects, plant parts, or printed letters. Remember: It’s the earliest type of microscope discovered by Antonie van Leeuwenhoek (in the late 1600s).

6. Compound Light Microscope Principle: Uses two sets of lenses — the objective lens and the eyepiece lens — to produce a much higher magnification. Construction: Objective lens: near the specimen; forms a real, enlarged image. Eyepiece lens: further enlarges that image. Light source: illuminates the specimen from below. Stage: where the specimen slide is placed. Mirror or lamp: directs light upward. Magnification: Up to about 1000× to 1500× . Resolution limit: About 0.2 micrometers (µm) – meaning objects closer than 0.2 µm appear as one. Used for: Viewing cells, bacteria, and tissues . Key Point: It uses visible light and glass lenses . Compound microscope was invented by Zacharias Janssen and his father Hans Janssen (around 1590 ).

7. Phase Contrast Microscope Principle: Some parts of a cell are transparent and colorless . Normally, we can’t see them clearly under a regular light microscope. The phase contrast microscope changes tiny differences in light phase (caused by varying densities of cell parts) into differences in brightness and contrast . Construction & Working: Uses special phase plates and annular diaphragms . Converts the invisible phase changes into visible contrast. Used for: Observing living, unstained cells (like amoeba, sperm, or dividing cells). Magnification: Similar to light microscope (up to ~1000×). 🟢 Key Point: It helps to study living cells without killing or staining them.

8. 1. Why was it invented? When we use a normal light microscope , living cells (like amoeba, white blood cells, or plant cells) often look almost transparent — it’s hard to see their internal structures. To make them visible, we usually stain them. But staining kills the cells, so we cannot watch them alive and moving . That’s a problem — especially if we want to study cell division, movement, or living processes . So, in 1934, a Dutch scientist named Frits Zernike invented the Phase Contrast Microscope , for which he won the Nobel Prize in 1953.

9. 2. Basic Principle To understand the principle, let’s recall something about light : When light passes through a specimen: Some parts of the specimen are dense (like nucleus, mitochondria), Other parts are less dense (like cytoplasm). These dense and less dense regions slow down the light differently — meaning the light waves come out with tiny phase differences (a small shift in the timing of light waves). Our eyes cannot detect these tiny phase differences — that’s why the image looks faint and colorless. The Phase Contrast Microscope converts these invisible phase differences into visible changes in brightness and contrast . So you can clearly see structures inside a living, unstained cell !

10. 3. How It Works (Simplified Steps) Let’s go step by step: Step 1️⃣: Light passes through the specimen The microscope uses a special condenser that sends light through the transparent specimen. Different parts of the specimen slow down the light differently (causing phase shifts). Step 2️⃣: Phase changes are introduced Light that passes through thick or dense parts of the specimen lags behind (phase retarded). Light passing through thinner areas moves faster (phase advanced). Step 3️⃣: Conversion to brightness difference Inside the microscope, there are special components called: Annular diaphragm (in the condenser) Phase plate (near the objective lens) These two parts work together to convert phase differences into intensity (brightness) differences . Step 4️⃣: The image is formed Now, the human eye (or camera) can see variations in brightness — dark and light areas — corresponding to different cell parts. Thus, transparent structures become visible without staining !

11. 4. Construction (Main Parts) Part Function Light Source Provides illumination. Annular Diaphragm Produces a hollow cone of light. Condenser Lens Focuses the light on the specimen. Phase Plate Converts phase differences into brightness differences. Objective and Eyepiece Lenses Magnify the image. Stage Holds the specimen.

12. 5. Advantages ✅ Can observe living cells without killing them. ✅ No need for staining . ✅ Excellent for studying motility, cell division, and cytoplasmic streaming . ✅ Simple to use once set up.

13. 6. Limitations ❌ Not suitable for very thick specimens (causes blurring). ❌ Produces halo effect (a bright ring around objects). ❌ More expensive than simple light microscopes. ❌ Needs special optical components .

14. 7. Applications Studying living protozoa (like Amoeba, Paramecium). Observing cell movement and mitosis . Viewing tissue culture cells. Examining bacterial motility . Used in medical and biological research .

15. Electron Microscope Principle: Instead of light, it uses a beam of electrons to form highly magnified images. Because the wavelength of electrons is much shorter than that of visible light, the resolution is extremely high . Types of Electron Microscopes: Transmission Electron Microscope (TEM) Electrons pass through the specimen. Gives a 2D image of internal structures. Resolution: up to 0.1 nanometers (nm) . Magnification: up to 10,00,000× . Used for: seeing cell organelles like mitochondria, ribosomes, membranes. Scanning Electron Microscope (SEM) Electrons scan the surface of the specimen. Gives a 3D image of surface structure. Used for: studying surface details of cells, pollen grains, insects, etc. Key Point: Electron microscopes need a vacuum and specimens must be dead and coated with metal .

16. 1. Why was it developed?

17. 2. Basic Principle

18. 3. How it Works — Step by Step

19. Step 2: Focusing the Beam

20. Step 3: Interaction with the Specimen

21. Step 4: Image Formation

22. 4. Types of Electron Microscopes Type Full Form What it shows Image Type Used For TEM Transmission Electron Microscope Internal structure (electrons pass through) 2D Cell organelles, viruses SEM Scanning Electron Microscope Surface structure (electrons bounce off) 3D Surfaces of cells, insects, pollen grains

23. 5. Magnification & Resolution

24. 6. Limitations ❌ Works only in vacuum , so living cells can’t be seen . ❌ Expensive and requires expert handling. ❌ Specimen preparation is complex — thin slicing, metal coating, dehydration.

25. 7. Advantages ✅ Extremely high resolution and magnification . ✅ Can show fine details of cell organelles, viruses, and molecular structures. ✅ Provides 2D (TEM) or 3D (SEM) images.

26. 8. In Short

27. Comparison Table Feature Simple Microscope Compound Microscope Phase Contrast Electron Microscope Source Visible light Visible light Visible light Electron beam Lenses 1 lens 2 or more lenses Special optical lenses Electromagnetic lenses Image type Virtual Real & virtual Contrast image TEM – 2D, SEM – 3D Max. magnification ~20× ~1500× ~1500× Up to 1,000,000× Living specimen Yes Yes Yes No Resolution Low Moderate Moderate Very high

28. Thank you