Observation Under the Microscope
Ximena scrapes a transparent layer off an onion from the market, puts it under the microscope at her secondary school, and discovers a grid of little compartments that can't be seen with the naked eye. Cells can't be seen without help, so you need an instrument that enlarges them hundreds of times. Using it well isn't automatic: it has its parts, its steps, and its tricks. And its history: about 350 years ago, nobody knew that cells existed.
Parts of the microscope
The one you use in your school lab is a compound optical microscope: "compound" because it combines two lenses (the eyepiece and the objective), and "optical" because it works with visible light. To see cells clearly, you need to hold the sample, light it, and focus it. Which parts do each of these jobs?
| Part | Function |
|---|---|
| Eyepiece | Lens you look through; it usually magnifies $10\times$. |
| Objectives | Lenses close to the sample ($4\times$, $10\times$, $40\times$); you switch them by turning the nosepiece. |
| Stage and clips | Hold the slide and let you move it. |
| Light source and diaphragm | Light the sample and control how much light passes through. |
| Arm and base | Provide stability and connect all the parts. |
| Coarse and fine focus knobs | Move up or down to focus: one coarsely and the other finely. |
In the light microscope diagram, there are three parts labelled with "?". Write the name of each one and explain its function.
In the light microscope diagram, there are three parts labelled with "?". Write the name of each one and explain its function.
Total magnification
When you look through the eyepiece, the image passes through two lenses: first the objective enlarges it, and then the eyepiece enlarges that already-enlarged image. That's why the magnifications are multiplied.
A microscope has an eyepiece lens of $10\times$ and objective lenses of $4\times$, $10\times$, and $40\times$. Calculate the total magnification achieved with each…
A microscope has an eyepiece lens of $10\times$ and objective lenses of $4\times$, $10\times$, and $40\times$. Calculate the total magnification achieved with each objective lens, and explain which one you should always start observing with and why.
Preparing a sample
The optical microscope works because light passes through what you're observing. That's why the sample must be very thin and transparent, and it is often stained: a dye highlights structures that are barely visible naturally. In the school lab, they use iodine solution (a brown or amber stain that highlights the nucleus of onion cells) and methylene blue (it colors the nucleus blue, for example in cheek cells).
Put the steps for preparing an onion cell sample in order: (a) cover with the coverslip taking care to avoid air bubbles, (b) place a thin layer of onion on the…
Put the steps for preparing an onion cell sample in order: (a) cover with the coverslip taking care to avoid air bubbles, (b) place a thin layer of onion on the microscope slide, (c) add a drop of iodine solution (lugol) to stain, (d) remove excess liquid with filter paper.
Why are many samples stained before observing them under a microscope? Give an example of a stain used in the school laboratory.
Why are many samples stained before observing them under a microscope? Give an example of a stain used in the school laboratory.
Focusing correctly
This is done for safety. At low magnification, the objective sits far from the preparation and there is room for error. At high magnification, the lens is almost touching the coverslip.
Why must you focus first using the lowest power objective lens and reserve the fine focus knob for higher power objective lenses? Explain what might happen if you use…
Why must you focus first using the lowest power objective lens and reserve the fine focus knob for higher power objective lenses? Explain what might happen if you use the coarse focus knob with the $40\times$ objective lens.
When the image looks bad
Sometimes you look through the eyepiece and the image doesn't help. Think about two questions: is enough light getting through? Is the sample where it should be?
| Problem | Possible causes | What to do (without changing objectives) |
|---|---|---|
| Very dark image | Diaphragm almost closed or light source at low intensity | Open the diaphragm and raise the light intensity |
| Very dark image | Sample too thick or too much dye | Prepare another sample that is thinner or with less stain |
| Only a fragment is visible | Sample poorly centered over the hole in the stage | Move the slide with the clips until it is centered |
| Only a fragment is visible | Small sample, with bubbles or not enough liquid | Prepare it again with the sample well spread out and without bubbles |
When observing a sample, the image looks very dark and only a fragment of the specimen can be seen. State two possible causes for each problem and what you would do to…
When observing a sample, the image looks very dark and only a fragment of the specimen can be seen. State two possible causes for each problem and what you would do to correct them without changing the objective lens.
The field of view
The field of view is the circle you see through the eyepiece. The more you magnify, the less of the sample fits in it. Each time the magnification is multiplied by a number, the diameter of the field is divided by that same number.
With the $10\times$ objective lens (total magnification $100\times$), the field of view is about $1.5\ \text{mm}$ in diameter. If an onion cell is about…
With the $10\times$ objective lens (total magnification $100\times$), the field of view is about $1.5\ \text{mm}$ in diameter. If an onion cell is about $0.25\ \text{mm}$ long, estimate how many cells in a row would fit across the field of view, and explain what will happen to the diameter of the field of view when switching to the $40\times$ objective lens.
From the first lenses to the electron microscope
Microscopes have changed enormously since the first observations.
| Era | Instrument and observation |
|---|---|
| 1600s | First microscopes with one or two lenses. In 1665 Robert Hooke observed a thin slice of cork and saw little compartments: he called them "cells." |
| 1670s | Antonie van Leeuwenhoek, with small, very well-polished lenses, saw protozoa and bacteria in water: it was the first time anyone had observed living microorganisms. |
| 1800s | Compound optical microscopes improve; cell theory is formulated and the role of bacteria in diseases is discovered. |
| 1930s onward | The electron microscope uses a beam of electrons instead of light and achieves magnifications of hundreds of thousands of times. For the first time, viruses and the inside of cells can be seen in detail. |
| Optical microscope | Electron microscope | |
|---|---|---|
| What it uses | Light and glass lenses | Beam of electrons |
| What is seen well | Cells, protozoa, bacteria (although small) | Organelles, bacteria in detail, and viruses |
| Sample | Can be alive | Dead, very thin, and in a vacuum |
| Image | In color (with staining) | In black, white, and grays |
Thanks to these instruments we know that bacteria are simple cells and that viruses are even smaller and are not cells.
A research team wants to film in real time how a unicellular protozoan swims and captures bacteria in a drop of stagnant water, and at the same time, photograph in full…
A research team wants to film in real time how a unicellular protozoan swims and captures bacteria in a drop of stagnant water, and at the same time, photograph in full detail the internal structure of the viruses that infect those bacteria. A student proposes using only a transmission electron microscope to achieve both goals. Evaluate their proposal with reasoned arguments: explain why it is not possible to observe the live protozoan with this device and determine which microscope should be used in each case and why.
What the microscope tells us about nutrition
Under the microscope you see that each cell is a living unit: it needs nutrients and oxygen, and it produces waste. A cell in your body can't go out to look for its food. Someone has to bring it. That's the job of nutrition: the set of processes by which the body gets from food the nutrients and oxygen it needs, transports them to all the cells, and eliminates the waste that is produced.
Four systems work in coordination: the digestive system (breaks down food and absorbs nutrients), the respiratory system (takes oxygen from the air and expels carbon dioxide), the circulatory system (the blood carries everything to the cells), and the excretory system (eliminates waste, such as that in urine).
During a practical activity observing cheek cell specimens stained with methylene blue, a group can only see shapeless blue smudges. Suggest at least three errors they…
During a practical activity observing cheek cell specimens stained with methylene blue, a group can only see shapeless blue smudges. Suggest at least three errors they might have made when preparing the sample and explain how you would repeat the experiment to get good results.
Summary
Go back to the beginning: Ximena's onion cells can be seen because light passes through a thin, stained, well-focused layer, and because two lenses multiply the magnification.