Light microscopy

Microscope Components and Their Functions

Have you ever wondered how we can see things smaller than a grain of sand? Light microscopes use a system of glass lenses to refract (bend) light, making tiny specimens appear much larger.

• Eyepiece Lens: The part you look through. It has a fixed magnification (e.g., x10) and further magnifies the image produced by the objective lens.
• Objective Lenses: Typically three or four lenses of different powers (e.g., x4, x10, x40). These are closest to the specimen and provide the primary magnification.
• Stage: The flat platform where the microscope slide is placed, held secure by stage clips.
• Lamp: Located at the base, it shines light upwards through the specimen so it can be seen clearly.
• Coarse Adjustment Knob: Rapidly moves the stage up and down to bring the specimen into rough focus. It should only be used with the lowest power objective lens.
• Fine Adjustment Knob: Moves the stage very slightly for precise focusing to produce a sharp, clear image.

The Purpose of Staining

If you looked at most living cells under a microscope, you wouldn't see much. Because biological cells have a high water content, they are transparent and virtually colourless.

To solve this, we use a stain. Stains are chemical dyes that bind to specific organelles, increasing contrast between the cell structures and the background. This allows detailed observation of specific parts of the cell.

• Iodine solution: Used for plant cells (e.g., onion epidermis). It reacts with starch to turn blue-black, highlighting starch granules, and tints the cell wall and nucleus brown.
• Methylene blue: A positively charged dye that binds to negatively charged nucleic acids (DNA and RNA). It is used for animal cells (e.g., cheek cells) to make the nucleus stand out in dark blue.
• Crystal violet: Used to highlight bacterial cell walls.

Preparing and Viewing Slides (OCR PAG 1)

To actually view these cells, you must prepare a slide carefully to avoid trapping air and creating artifacts.

A slide is a clear rectangular piece of glass that holds the specimen. A cover slip is a small, thin square of glass placed over the specimen to keep it flat, hold it in place, and protect the objective lens from moisture.

Preparing an Onion Cell Slide (Plant):

1. Peel a thin, transparent layer of epidermal tissue from the inner onion using forceps.
2. Place the tissue flat on a clean slide, ensuring no folds.
3. Add two drops of iodine solution.
4. Lower the cover slip at an angle using a mounted needle to prevent trapping air bubbles.

Preparing a Cheek Cell Slide (Animal):

1. Gently scrape the inside of the cheek with a sterile cotton swab.
2. Smear the swab onto the centre of a slide and add a drop of methylene blue.
3. Lower the cover slip. Immediately place the cotton swab in disinfectant.

Note: If a slide is already prepared, you can use the irrigation technique. Place a drop of stain on one edge of the cover slip and a paper towel on the opposite edge to draw the stain across the specimen.

Viewing the Specimen:

1. Place the slide on the stage and secure it with clips.
2. Select the lowest power objective lens (this provides a larger field of view and prevents the lens from crashing into the slide).
3. Looking from the side, use the coarse adjustment knob to move the stage up to just below the lens.
4. Look through the eyepiece and use the coarse adjustment to move the stage away from the lens until roughly in focus.
5. Use the fine adjustment knob to get a clear, sharp image.

Magnification Calculations

A magnified image is only useful if we know its true scale. We measure this using two concepts: magnification (how much larger the image is than the real object) and resolution (the ability to distinguish between two close points).

Magnification is a ratio and has no units, but is written with an 'x' (e.g., x400). To calculate it, all measurements must be in the same units.

Unit Conversions:

• $1 \text{ cm} = 10 \text{ mm}$
• $1 \text{ mm} = 1,000 \text{ }\mu\text{m}$ (micrometres)
• $1 \text{ }\mu\text{m} = 1,000 \text{ nm}$ (nanometres)

The formula for magnification is:

$$M = \frac{I}{A}$$

Where:

• $M$ = Magnification
• $I$ = Image size (how big it appears on paper)
• $A$ = Actual size (the real-life size of the specimen)

Worked Example: Calculating Magnification

Question: A cell measures $40 \text{ mm}$ on a printed image. The actual size of the cell is $20 \text{ }\mu\text{m}$. Calculate the magnification.

Step 1: Convert units so they match (convert mm to $\mu m$).

• $40 \text{ mm} \times 1,000 = 40,000 \text{ }\mu\text{m}$

Step 2: Substitute into the formula ($M = I \div A$).

• $M = 40,000 \div 20$

Step 3: Calculate the final answer.

• $M = 2,000$
• Answer: x2,000 magnification.