Cell microscopy and estimating sub-cellular size

To prepare an onion slide, use tweezers to peel a thin layer of tissue from an onion layer. Place it flat on a slide without folds, and add a drop of iodine. Carefully lower a coverslip at an angle to avoid trapping air bubbles. The coverslip keeps the specimen flat and retains liquid underneath it. Blot away excess liquid with a paper towel. A thin, flat specimen lets light pass through without several layers of cells obscuring each other.

Finding and focusing the cells

The stage supports the slide, and clips hold it in position. The objective lenses, mounted on a rotating nosepiece, provide different magnifications. You look through the eyepiece lens. Coarse adjustment makes larger focusing movements; fine adjustment makes smaller movements that sharpen the image.

Use a light microscope, plant and animal slides, and a pencil and ruler for recording your observations.

1. Place the slide on the stage and secure it with the clips. Arrange the light source or mirror so that light passes through the specimen.
2. Select the lowest-power objective, usually ×4. This gives a wide field of view, making cells easier to find and showing their arrangement.
3. Looking from the side, use coarse adjustment to bring the objective close to the slide without touching it. Depending on the microscope, the stage or the lens moves.
4. Look through the eyepiece and slowly increase the distance between the objective and slide with coarse adjustment until the cells come into focus.
5. Use fine adjustment to sharpen the image. Find a suitable group of cells and centre it in the field of view.
6. Rotate the nosepiece to a higher-power objective, then use fine adjustment to refocus.

Higher power enlarges individual cells but shows a smaller area of the specimen. Repeat the observations with both plant and animal specimens.

Recording what you observe

On an onion slide, look for the cell walls outlining neighbouring cells and, where clearly stained, nuclei and cytoplasm. On a cheek-cell slide, look for each cell's boundary, cytoplasm and stained nucleus. The exact structures visible depend on the specimen, staining and focus. Ribosomes are too small to distinguish with a school light microscope; the absence of visible detail does not mean a structure is absent from the cell.

Make a large, clear pencil drawing of a few representative cells. Use continuous outlines without shading or sketchy strokes, and keep the structures in proportion. Draw straight label lines with a ruler, touching the structures they identify and avoiding crossed lines. Give the drawing a title identifying the specimen.

Record the microscope magnification underneath:

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

For example, a ×10 eyepiece with a ×4 objective gives ×40 viewing magnification. With a ×40 objective, the same eyepiece gives ×400.

A scale bar gives a direct indication of actual size. Its length must match the scale of the particular image. For example, in an image enlarged ×1000, a 10 mm bar represents 10 µm in the specimen: 10 µm is 0.01 mm, and $0.01 \times 1000 = 10$. Label the bar 10 µm, because that is the actual distance represented. Do not attach an arbitrary scale bar to a drawing whose scale is unknown.

Estimating relative lengths

Estimation is useful when you need an approximate comparison rather than an exact measurement. Cell boundaries may be irregular or unclear, and structures vary between cells, so reporting a highly precise value can be misleading.

Compare structures within the same image, or images at the same magnification. Measure the same kind of dimension for both: for example, width across the nucleus and width across the whole cell.

In one image, suppose a cell measures about 24 mm across and its nucleus about 6 mm across. Dividing $24 \div 6 = 4$ shows that the cell is about four times as wide as its nucleus. The actual dimensions are not needed for this relative comparison, because both image measurements have been enlarged by the same factor.

Estimating relative areas

Area is the amount of a two-dimensional image occupied by a structure. It is different from length, so a width comparison alone does not establish an area comparison.

One practical way to estimate area is to place a square grid over an image. Count complete squares within each boundary, then combine partly covered squares into approximate whole squares. Use the same grid for both structures.

For one cell image, imagine that the whole cell covers about 40 squares and its nucleus covers about 8. The nucleus occupies about $8/40 = 1/5$ of the cell's image area. If the remaining area is cytoplasm, its estimated area is $40 - 8 = 32$ squares, giving a cytoplasm-to-nucleus area ratio of $32:8$, or $$.

These are estimates of the areas visible in the image, not measurements of the structures' three-dimensional volumes. Clearer images and more careful boundary measurements are needed when a precise value matters.

• Observe a selection of plant and animal cells; draw and label visible structures and include a magnification scale.
• Thin, flat specimen; stain for contrast; lower the coverslip carefully to avoid air bubbles.
• Iodine: onion cells. Methylene blue: cheek cells.
• Start on low power: wider field of view, easier to locate cells.
• Focus with coarse adjustment, then fine adjustment. After increasing power, refocus with fine adjustment.
• Pencil drawing: clear outlines, correct proportions, no shading, straight label lines.

Magnification and scale

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

Record viewing magnification. A scale bar shows an actual specimen distance and must match the image's scale.