Light microscopy

Why cells need to be magnified

Most cells are too small to examine clearly with the unaided eye. A light microscope passes visible light through a thin specimen and uses lenses to produce a larger image. This lets us observe the shapes and arrangements of cells and some of their internal structures.

Making an image larger is not the same as revealing unlimited detail. Magnification tells us how many times larger the image is than the specimen, whereas resolution describes whether two very close points can be distinguished. An image can therefore be highly magnified but still lack fine detail if the microscope cannot resolve it.

How the microscope forms an image

The lamp provides the light. The light travels upwards through the specimen on the slide and then enters the lenses.

The specimen is first magnified by an objective lens, which is positioned close to the slide. The eyepiece lens then magnifies this image again for the observer. Microscopes usually have several objective lenses so that different magnifications can be selected.

The stage supports the microscope slide and holds it in the path of the light. The specimen must be thin enough for light to pass through it. A thick or folded specimen may appear dark, contain overlapping cells or be difficult to focus.

Preparing a specimen on a slide

A small, thin specimen is placed flat on a clean glass slide. For example, a thin layer of onion epidermis can provide a sheet of plant cells. A suitable liquid or stain is added, and a cover slip is lowered gently over the specimen.

The cover slip keeps the specimen flat and creates a thin layer through which light can pass. Lowering it slowly at an angle helps to push air out rather than trapping bubbles. An air bubble can look like part of the specimen and may obscure cells.

The slide is then placed on the stage above the lamp. It should be secured and positioned so that the specimen lies over the opening through which the light passes.

Why stains are useful

Many cells and cell structures are nearly colourless. Light may pass through them without producing enough contrast between neighbouring areas, so their outlines can be difficult to distinguish.

A stain adds colour and increases contrast. It may make the whole specimen easier to see or highlight particular structures or tissues more strongly than others. For example, iodine solution can be used when preparing onion epidermis cells. Staining changes the visibility of structures; it does not increase the microscope's magnification.

Focusing the microscope

Start with the lowest-power objective lens. This gives the widest field of view, so the specimen is easier to find.

Use the coarse-focus control to bring the specimen into general focus, then use the fine-focus control to sharpen the image. Centre the area of interest before selecting a higher-power objective, because increasing magnification reduces the field of view. At higher power, refocus carefully using the fine-focus control.

The image seen through a light microscope is a flat view of a thin section or layer. The original cell is three-dimensional, so changing the focus may bring different depths of the specimen into view.

Calculating total microscope magnification

The objective lens magnifies the specimen and the eyepiece magnifies that image, so their magnifications are multiplied:

$\text{total microscope magnification}=\text{eyepiece magnification}\times\text{objective magnification}$

Magnification is written using the multiplication sign, such as $\times 200$. It has no measurement unit because it is a ratio.

Calculating magnification from an image

For a biological image or micrograph:

$\text{magnification}=\frac{\text{image size}}{\text{actual size}}$

The image size and actual size must be expressed in the same unit before division. A useful conversion is:

$1\text{ mm}=1000\text{ µm}$

The relationship can also be rearranged:

$\text{actual size}=\frac{\text{image size}}{\text{magnification}}$

$\text{image size}=\text{magnification}\times\text{actual size}$

If an image has a magnification of $\times 200$, every measured length in the image is 200 times the corresponding actual length. Magnification refers to linear size, such as width or length—not area or volume. When measuring a printed or displayed image, use its current measured size because resizing the image changes its magnification.