Microscope Bench · MicroLab

Meet the microscope

A light microscope consists of an optical system and a mechanical system. The optical parts are the eyepiece, objectives and mirror (or light source); the mechanical parts include the base, pillar, arm, body tube, revolving nosepiece, stage and focus knobs.

Hover over any part of the microscope to see its name and function. Turn on the exploded view to see how the body tube and objectives connect.

Magnification = eyepiece power × objective power

Objectives have a screw thread: the longer, the higher the power. Eyepieces have no thread: the longer, the lower the power. The colour ring on an objective shows its power: red 4×, yellow 10×, blue 40×, white 100× (oil immersion).

Proper technique

Use the microscope following the textbook steps. The checklist below ticks off each step, and any mistakes are logged in the data dock.

1. Carrying and placing: grip the arm with your right hand and support the base with your left; set it on the bench slightly to the left.

2. Adjusting the light: turn the nosepiece so the low-power objective is over the stage aperture; line up a large opening of the diaphragm with the aperture; looking into the eyepiece with your left eye, turn the mirror until you see a bright, round field.

3. Observing: put the slide on the stage with the specimen centred over the aperture and hold it with the stage clips. Turn the coarse focus knob to lower the tube slowly, watching the objective from the side, until it is close to the slide; then look into the eyepiece and turn the coarse knob the other way to raise the tube slowly until the image appears, and finally turn the fine focus knob slightly to sharpen it.

Image formation

Write a tiny letter “e” on a slide and put it under the microscope, and you see “ə” instead — flipped both top-to-bottom and left-to-right. A microscope forms an inverted image, as if the slide were rotated 180° on the page.

In this lesson, dragging in the field is set to “push the slide”: whichever way your hand pushes the slide, the image moves the opposite way.

Image is off to the upper left → move the slide toward the upper left and the image returns to the centre

Try again with the slide of the character meaning “up”, and compare the image in the field with the slide in the 3D scene.

Low to high power

The field of view is small under high power, so you must first find the target under low power, move it to the center of the field, and then turn the revolving nosepiece to the high-power objective.

After switching to high power:

· The field becomes smaller and darker: open the diaphragm or switch to the concave mirror;· Depth of field becomes shallower: use only the fine focus knob — a single click of the coarse focus knob may crush the slide;· You see fewer cells, and each cell looks larger.

Field diameter = field number ÷ objective magnification Brightness ∝ (NA ÷ objective magnification)²

The target cell is circled with a dashed line. The "Imaging parameters" table in the data dock compares the four objectives.

Locate the speck

There is a speck in the field of view. It may be on the eyepiece, the objective or the slide.

· Rotate the eyepiece: the speck turns with it → on the eyepiece;· Move the slide: the speck moves with it → on the slide;· Neither moves it, and it disappears when you change objectives → on the objective.

Observe first, then answer in the data dock. Press "New speck" to practice more.

Plant cell

Peel off the inner epidermis of an onion bulb scale, spread it flat in a drop of water and add a coverslip. Unstained cells are almost transparent and only the cell walls are clear — try closing the diaphragm: transparent structures will show their outlines.

Click the iodine solution on the bench and add it at one edge of the coverslip, then click the filter paper to draw it from the other edge (wicking). The iodine gradually flows across the specimen: nuclei stain dark brown and nucleoli become clearly visible.

Basic structures of a plant cell: cell wall, cell membrane, cytoplasm, nucleus, vacuole. Onion epidermal cells have one large central vacuole, which often pushes the nucleus to the edge of the cell.

Turn the fine focus knob at 40×: cells are about 25 µm thick, and you see different structures when the focal plane is at the top surface, the middle or the bottom surface.

Animal cell

Gently scrape the inside of the cheek with a sterile toothpick and smear it on a slide with a drop of physiological saline (in plain water the cells absorb water and burst), then add a coverslip.

Cheek epithelial cells are flat and irregular in shape, often clumped and overlapping, with folded edges; the nucleus is small and central. They are so thin they are almost invisible unstained; after staining with methylene blue the nuclei turn dark blue. Oral bacteria are often attached to the cell surface.

Compared with onion epidermal cells: animal cells have no cell wall and no large vacuole, and their shape is not fixed.

Paramecium

Take a drop from the surface layer of a Paramecium culture (more oxygen there, so more paramecia), add a few cotton fibers to slow them down, and add a coverslip.

Paramecium is a single-celled organism shaped like an upside-down slipper, about 200 µm long. Its surface is covered with cilia, whose beating makes it spin forward; when it hits an obstacle it backs up, turns and moves forward again.

At 10× you can see: the oral groove, food vacuoles (carried by the flowing cytoplasm), two contractile vacuoles (with radiating collecting canals, contracting regularly to expel excess water), and the macronucleus and micronucleus.

They swim fast — add cotton fibers and track them under low power.

Leaves and chloroplasts

The small leaves of Hydrilla are only one or two cell layers thick and can be mounted directly. The chloroplasts in the cells are flattened ellipsoids that flow along the cell edge around the central vacuole — this is cytoplasmic streaming. Using the chloroplasts as markers, you can tell the direction of flow.

Switch to the broad bean leaf lower epidermis slide: the epidermal cells interlock like jigsaw pieces; pairs of crescent-shaped guard cells surround each stoma. Guard cells contain chloroplasts; ordinary epidermal cells do not.

In the leaf cross-section slide you can see the upper and lower epidermis, palisade tissue, spongy tissue and veins.

Plasmolysis and deplasmolysis

The vacuoles of purple onion bulb scale outer epidermis cells contain anthocyanin, which makes the position of the protoplast layer easy to see.

Add 0.3 g/mL sucrose solution at one edge of the coverslip and draw it through with filter paper from the other edge. The outside solution is more concentrated than the cell sap, so the cells lose water and the protoplast layer (cell membrane, tonoplast and the cytoplasm between them) gradually separates from the cell wall — starting at the corners; the vacuole shrinks and the purple deepens.

Then add water in the same way: the cells take up water and deplasmolysis occurs.

Elasticity of the protoplast layer > elasticity of the cell wall

The data dock records how the average protoplast area of cells in the field changes over time.

Mitosis

Onion root tips go through dissociation (hydrochloric acid + alcohol, to separate the cells) → rinsing → staining (crystal violet solution, to color the chromosomes) → mounting (squashing to spread the cells).

First find the meristematic zone under low power: the cells are square, tightly packed, and many are dividing; cells of the root cap and elongation zone look different.

After switching to high power, click a cell and decide which stage it is in: interphase, prophase, metaphase, anaphase or telophase. The data dock counts cells in each stage and calculates the mitotic index.

Ratio of cell numbers in each stage ≈ ratio of time spent in each stage

Note: the cells are dead after dissociation, so you see static cells — you cannot watch one cell divide continuously.

Blood smears and oil immersion

The most numerous cells in a human blood smear (Wright stain) are red blood cells: biconcave discs, thinner and paler in the center, with no nucleus. White blood cells are few and have nuclei: neutrophils have nuclei with 2~5 lobes, lymphocytes have large round nuclei, monocytes are the largest with kidney-shaped nuclei, and eosinophils contain orange-red granules. Platelets are tiny and have no nucleus.

The 100× objective is an oil immersion lens: its numerical aperture is 1.25, and you must put a drop of cedarwood oil on the coverslip, otherwise light is refracted and totally reflected at the glass–air interface and the image is blurry. Its working distance is only 0.15 mm.

Compare with a frog blood smear: frog red blood cells are oval and have nuclei.

Micrometer

The ocular micrometer sits inside the eyepiece; its scale is magnified with the eyepiece, and the real length each division represents changes with the objective, so it must be calibrated first. The stage micrometer is a slide with a precise scale: 1 mm divided into 100 divisions, 10 µm each.

Rotate the eyepiece so the two scales are parallel, move the stage micrometer so both 0 marks line up, then look to the right for the place where the two scales coincide again:

Length per eyepiece division = stage divisions × 10 µm ÷ eyepiece divisions

Enter the readings in the data dock and calibrate. Then switch to a cell slide (do not change the objective) and drag a line in the field to measure cell length.

Hemocytometer

The counting chamber of the hemocytometer is a 1 mm × 1 mm grid, 0.1 mm deep, divided into 25 medium squares of 16 small squares each.

Put on the coverslip first, then add yeast culture at the edge of the coverslip and let it seep in by itself. Wait a moment for the yeast to settle to the bottom of the chamber before counting.

Count the yeast in the 5 medium squares at the four corners and the center (80 small squares in all). Click a yeast cell to count it. For cells on the grid lines, count the top and left lines, not the bottom and right; a budding yeast counts as two only when the bud is at least half the size of the mother cell.

Yeast per mL = count in 80 small squares ÷ 80 × 400 × 10⁴ × dilution factor

Bright field, dark field and phase contrast

Most living cells are transparent: they only change the phase of light, not its brightness, so they are hard to see in bright field.

· Bright field: closing the aperture diaphragm raises contrast but lowers resolution; slight defocus gives transparent structures bright or dark edges.· Dark field: blocks direct light and lets only light scattered by the specimen enter the objective — cell outlines and tiny particles glow on a black background.· Phase contrast: a phase plate converts optical path differences into brightness differences, so transparent cells show clearly, with bright halos at their edges.

Resolution d = λ ÷ (NA objective + NA condenser)

Switch to diatom, Paramecium and other slides and compare the three methods.

Free observation

All slides and features are unlocked: take any slide from the slide tray and use any objective, eyepiece, illumination mode and reagent.

Tips:

· Scroll wheel in the field = fine focus, Shift + scroll = coarse focus;· Arrow keys move the slide, number keys 1~4 change objectives;· V enlarges the eyepiece view, S shows a side view of the objective;· With structure labels on, hover over structures in the field to see their names.