Introduction to Microscopes and Biological Measurements

Illumination and Focus

• Condenser: A lens system that focuses light into a beam of parallel waves onto the specimen.
• Iris Diaphragm: A mechanism that regulates the amount of light passing through the condenser. Reducing the light can actually increase the visibility of semi-transparent objects by improving contrast.
• Mechanical Stage: The flat surface that supports the slide. In compound microscopes, this stage often includes mechanical stage knobs to move the slide precisely.
• Focus Knobs: The coarse focus knob is used for initial, rough adjustments to the distance between the lens and the specimen. The fine focus knob is used for precise, sharp adjustments, especially at higher magnifications.

Exam Tip: Total Magnification To find the total magnification of your view, always multiply the magnification of the ocular lens by the magnification of the objective lens currently in use. $\text{Total Magnification} = \text{Ocular Magnification} \times \text{Objective Magnification}$

Optical Principles: Resolution, Magnification, and Depth of Field

Understanding the physics of light is essential for interpreting what you see through the lens.

Resolution

Resolution is the ability to distinguish between two points as separate entities. A high-resolution image is sharp, whereas a low-resolution image is fuzzy, regardless of how much it is magnified. Resolution can be improved by:

1. Using a blue filter: Shorter wavelengths of light (like blue) reduce diffraction and increase resolution.
2. Immersion Oil: Using a special oil with a $100\times$ objective lens decreases light diffraction, allowing more light to enter the lens.

Depth of Field

The depth of field refers to the vertical thickness of the specimen that remains in focus at a single time. There is an inverse relationship between magnification and depth of field: as magnification increases, the depth of field decreases.

Biological Measurements

Microscopy allows for the quantification of biological specimens through several mathematical approaches.

1. Estimating Specimen Size

You can calculate the actual size of a specimen by comparing how much of the "field of view" (the bright circle of light) it occupies.

Example Calculation: If a specimen occupies one-quarter ($1/4$) of the medium power field, and the field diameter is $2\text{ mm}$, what is the actual size of the specimen in micrometers ($\mu\text{m}$)?

$\begin{aligned} \text{Actual size} &= \text{fraction of field occupied} \times \text{field diameter} \\ &= \frac{1}{4} \times 2\text{ mm} \\ &= 0.5\text{ mm} \end{aligned}$

To convert to micrometers: $0.5\text{ mm} \times 1000\text{ }\mu\text{m}/\text{mm} = 500\text{ }\mu\text{m}$

2. Calculating Drawing Magnification

When creating scientific illustrations, you must indicate how much you have enlarged the specimen from its real size.

$\text{Magnification of drawing} = \frac{\text{size of drawing (mm)}}{\text{actual size of specimen (mm)}}$

3. Speed of Movement

If a specimen is moving, you can calculate its speed by measuring the distance it travels across the field over a specific time interval.

Worked Example: Speed A specimen moves across a medium power field ($1.8\text{ mm}$) in $20\text{ seconds}$. $\text{Speed} = \frac{1.8\text{ mm}}{20\text{ s}} = 0.09\text{ mm/s}$ To find mm per minute: $0.09\text{ mm/s} \times 60\text{ s/min} = 5.4\text{ mm/min}$