3. Using a Light Microscope

Using a Light Microscope

A light microscope uses visible light and a system of lenses to produce a magnified image of a small specimen.

Light microscopes allow scientists to observe structures that are too small to see clearly with the unaided eye, including:

• cells
• tissues
• microorganisms
• small parts of plants and animals

Using a microscope effectively involves more than simply looking through the eyepiece. The specimen must be prepared correctly, the image must be focused carefully, and observations must be recorded accurately.

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Important Parts of a Light Microscope

Before using a microscope, it is important to understand the main parts.

A typical eyepiece magnifies the image by 10x, while objective lenses commonly provide 4x, 10x, or 40x magnification.

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Preparing a Biological Specimen

Many biological specimens need to be placed on a microscope slide before they can be observed.

A common method is called a wet mount.

You will usually need:

• a clean microscope slide
• the specimen
• a drop of water
• a coverslip
• a dropper
• forceps or a mounted needle

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Preparing a Wet Mount

A simple wet mount can be prepared using the following procedure:

1. Place a thin specimen in the centre of a clean slide.
2. Add a small drop of water.
3. Hold a coverslip at an angle so that one edge touches the water.
4. Lower the coverslip slowly over the specimen.
5. Remove excess liquid with tissue if necessary.

Lowering the coverslip at an angle helps reduce the number of air bubbles trapped underneath it.

Air bubbles can be mistaken for biological structures and may make observations more difficult.

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Why Must the Specimen Be Thin?

Light needs to pass through the specimen before reaching the objective lens.

If the specimen is too thick:

• less light may pass through it
• cells may overlap
• individual structures may be difficult to distinguish

A thin specimen usually produces a clearer image.

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Using Stains

Many cells are almost transparent.

A stain can be added to increase contrast and make particular structures easier to see.

For example:

• iodine solution can improve the visibility of structures in some plant tissues
• methylene blue may be used with certain animal cells

Stains do not increase magnification. They make structures easier to distinguish by increasing contrast.

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Setting Up the Microscope

Before placing the specimen under high magnification, the microscope should be set up correctly.

Start by:

• placing the microscope on a stable, flat surface
• turning on the light
• rotating the lowest-power objective into position
• placing the slide on the stage
• securing it with the stage clips
• positioning the specimen over the opening in the stage

Starting with the lowest-power objective makes it much easier to locate the specimen.

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Why Start on Low Power?

Low magnification provides a:

• larger field of view
• brighter image
• greater working distance
• easier way to locate the specimen

For example, with a 4x objective, you can see a much larger area than with a 40x objective.

Once the specimen is located and centred, you can move to higher magnification.

Notice that as magnification increases, the field of view becomes smaller.

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Focusing on Low Power

With the lowest-power objective selected:

1. Look from the side and make sure the objective is not touching the slide.
2. Look through the eyepiece.
3. Use the coarse focus knob to bring the specimen into view.
4. Use the fine focus knob to sharpen the image.
5. Centre the part of the specimen you want to examine.

The coarse focus knob makes relatively large changes in the distance between the objective and specimen.

The fine focus knob makes much smaller changes.

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Moving to Higher Magnification

Once the specimen is clearly focused and centred:

1. Rotate the nosepiece to the next objective.
2. Look through the eyepiece.
3. Use the fine focus knob to sharpen the image.
4. Adjust the light if necessary.

At high power, avoid large movements using the coarse focus knob because the objective lens may be very close to the slide.

This can damage:

• the slide
• the specimen
• the objective lens

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Coarse Focus vs Fine Focus

Coarse Focus

Used mainly at low magnification.

It:

• moves the stage or objective a relatively large distance
• helps locate the specimen quickly
• brings the image roughly into focus

Fine Focus

Used for precise focusing.

It:

• moves the stage or objective only slightly
• sharpens details
• is especially important at high magnification

A simple rule is:

Lower power: course then fine

High popwer: fine focus

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Calculating Total Magnification

The total magnification depends on both the:

• eyepiece lens
• objective lens

Calculate total magnification using:

Total magnification = eyepiece magnification x objective magnification

For example, if the eyepiece is: 10x and the objective is 4x then:

10x40 = 40

So the total magnification is: 40x

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Worked Example

A microscope has a 10x eyepiece and a 40x objective.

Calculate the total magnification.

total magnification = 10x40

400x

The specimen appears approximately 400 times larger than when viewed without magnification.

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Common Magnifications

With a 10x eyepiece:

Higher magnification allows smaller structures to appear larger, but it also reduces the area visible at one time.

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Magnification Is Not the Same as Resolution

Magnification describes how much larger an image appears.

Resolution describes the ability to distinguish two nearby objects as separate.

A highly magnified image is not necessarily useful if it is blurry.

Good microscopy therefore depends on both:

magnification + resolution

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Adjusting Image Quality

Several microscope settings can be adjusted to improve the image.

The most important are:

• focus
• light intensity
• diaphragm opening
• specimen position
• magnification

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Adjusting the Light

Too little light can make the specimen difficult to see.

Too much light can wash out details and reduce contrast.

The diaphragm controls how much light passes through the specimen.

If the image appears too dark:

• increase the light intensity
• open the diaphragm slightly

If the image appears too bright:

• decrease the light intensity
• close the diaphragm slightly

The goal is not simply to make the image as bright as possible. The goal is to produce the clearest image with good contrast.

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Centring the Specimen

Before increasing magnification, place the object of interest near the centre of the field of view.

This is important because the field of view becomes smaller at higher magnification.

If the specimen is near the edge at low power, it may disappear completely when you switch to high power.

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Field of View

The field of view is the circular area visible through the microscope.

At low magnification: large field of view

At high magnification: small field of view

This means increasing magnification allows you to see more detail but a smaller area of the specimen.

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Brightness and Magnification

Images often appear darker when you increase magnification.

This happens because less light may reach your eye through the higher-power objective.

You may therefore need to:

• increase the light intensity
• adjust the diaphragm

whenever you move to a higher magnification.

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Moving the Slide

One unusual feature of a compound light microscope is that the image appears reversed.

If you move the slide: →

the image may appear to move: ←

Similarly, moving the slide upward can make the image appear to move downward.

This takes practice when trying to centre a specimen.

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Observing Biological Specimens

When viewing a specimen, do more than simply identify it.

Look carefully for:

• shape
• size
• arrangement
• colour
• boundaries
• visible internal structures
• differences between cells

For example, when viewing plant cells, you might observe:

• cell walls
• regular cell shapes
• nuclei if stained
• chloroplasts in suitable tissues

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Recording Observations

Scientific observations should be recorded accurately.

One common method is a biological drawing.

A biological drawing should show what you actually observe rather than what you expect to see.

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Making a Good Biological Drawing

A good microscope drawing should:

• be large enough to show detail
• use clear, single pencil lines
• avoid sketching or shading unless required
• show correct proportions
• include labels
• use straight label lines
• include a title
• state the magnification or scale when appropriate

Do not add structures that you could not actually see through the microscope.

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Example Observation Record

Suppose you observe onion epidermal cells.

A suitable record might include:

Specimen: Onion epidermis

Total magnification: 100x

Observations:

• cells are arranged closely together
• cells have rectangular shapes
• clear cell walls are visible
• some nuclei are visible after staining

A labelled biological drawing could then be added.

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Recording Magnification

Always record the magnification used for an observation.

For example: Magnification = 100x

This makes the observation more scientifically useful because another person can understand the scale at which the specimen was viewed.

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Observations vs Interpretations

It is important to distinguish an observation from an interpretation.

An observation describes what you actually see.

For example:

Most cells appear rectangular.

An interpretation explains what you think the observation means.

For example:

The rectangular shape is caused by the rigid cell wall.

Both can be useful, but they are not the same.

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A Complete Microscope Procedure

A reliable microscope procedure can be summarized as follows:

1. Carry the microscope safely to the work area.
2. Place it on a stable surface.
3. Turn on the light.
4. Select the lowest-power objective.
5. Prepare and position the slide.
6. Secure the slide on the stage.
7. Centre the specimen over the light.
8. Use coarse focus to locate the specimen.
9. Use fine focus to sharpen the image.
10. Adjust the diaphragm and light intensity.
11. Centre the region of interest.
12. Move to a higher-power objective if necessary.
13. Use fine focus to sharpen the high-power image.
14. Record observations and magnification.
15. Return to low power when finished.
16. Remove and clean the slide.
17. Turn off and store the microscope correctly.

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Microscope Safety

A microscope is a precision instrument and should be handled carefully.

Always carry it with two hands:

• one hand holding the arm
• one hand supporting the base

This reduces the risk of dropping it.

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Safe Use of the Objective Lenses

Never force an objective lens into the slide.

When bringing an objective close to a specimen:

• look from the side
• make sure there is enough space
• move slowly

At high power, use the fine focus knob rather than making large coarse-focus adjustments.

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Caring for the Lenses

Microscope lenses can be easily scratched.

Only use appropriate lens paper or approved lens-cleaning material to clean them.

Do not use:

• paper towels
• clothing
• tissues intended for general cleaning
• your fingers

Fingerprints and scratches can reduce image quality.

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Handling Slides

Glass slides and coverslips can break and produce sharp edges.

Handle them carefully.

If glass breaks:

• do not pick it up with bare hands
• tell the teacher or laboratory supervisor
• dispose of it using the correct broken-glass procedure

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Common Microscope Problems

I Cannot See the Specimen

Possible reasons:

• specimen is not centred
• wrong objective is selected
• image is badly out of focus
• slide is upside down
• light is not passing through the specimen

Return to low power, centre the specimen, and refocus.

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The Image Is Too Dark

Try:

• increasing light intensity
• opening the diaphragm
• checking that the specimen is over the light opening

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The Image Is Too Bright

Try:

• reducing light intensity
• closing the diaphragm slightly

Too much light can reduce contrast.

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The Image Is Blurry

Try:

• adjusting fine focus
• checking that the coverslip is flat
• cleaning the lenses correctly
• checking whether the specimen is too thick

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The Specimen Disappears on High Power

This usually happens because it was not centred before changing objectives.

Return to low power:

1. locate the specimen again
2. centre it
3. refocus
4. switch to high power

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Worked Example: Choosing the Correct Magnification

A student wants to locate a small group of cheek cells and then examine one cell closely.

The best procedure is:

First: low magnification

because it provides a larger field of view.

Once the cells are located and centred, switch to: higher magnification to see more detail.

Starting immediately at high power would make finding the cells much more difficult.

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Worked Example: Improving Image Quality

A student can see a cell, but the image is extremely bright and the structures are difficult to distinguish.

The student should:

• reduce the light intensity or
• partially close the diaphragm
• then use the fine focus knob

The problem is not necessarily insufficient magnification. It is poor contrast and focus.

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Comparing Low and High Magnification

This is why good microscope technique always begins at low magnification.

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Effective Microscope Technique

A skilled microscope user develops a consistent routine:

Locate → Focus → Center → Magnify → Refocus

Skipping one of these steps often makes microscopy more difficult.

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Common Misconceptions

Higher magnification is always better.

Not necessarily. Low magnification is better for locating specimens and viewing larger structures.

The coarse focus knob should be used at every magnification.

No. At high power, large adjustments can cause the objective to strike the slide.

More light always produces a better image.

Too much light can reduce contrast and make structures harder to distinguish.

Magnification and resolution are the same thing.

Magnification makes the image larger. Resolution determines how clearly nearby structures can be distinguished.

Scientific drawings should show everything known about the cell.

They should show what was actually observed.

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Did You Know?

The image produced by a compound light microscope is typically inverted relative to the specimen.

This means that when you move the slide in one direction, the image appears to move in the opposite direction.

Microscopy also involves a trade-off: increasing magnification usually decreases the field of view, which is why experienced microscope users always locate and centre specimens before switching to high power.

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Key Terms

Light microscope – An instrument that uses visible light and lenses to magnify a specimen.

Specimen – The material being observed.

Slide – A thin piece of glass used to support a specimen.

Coverslip – A thin piece of glass placed over a specimen.

Wet mount – A slide preparation in which a specimen is placed in a drop of liquid.

Magnification – How many times larger an image appears.

Resolution – The ability to distinguish two nearby objects as separate.

Field of view – The area visible through the microscope.

Coarse focus – A control that makes large focusing adjustments.

Fine focus – A control that makes small, precise focusing adjustments.

Diaphragm – A part of the microscope that regulates the amount of light passing through the specimen.

Contrast – The difference between light and dark areas that allows structures to be distinguished.

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Key Takeaways

• A light microscope allows us to observe cells and other small biological structures.
• Biological specimens should be prepared carefully, often using a wet mount.
• Begin observation using the lowest-power objective.
• At low power, use the coarse focus followed by the fine focus.
• At high power, use the fine focus for precise adjustments.
• Total magnification is calculated using: eyepiece magnification x oobjective magnification
• Increasing magnification decreases the field of view.
• Image quality can be improved by adjusting focus, light intensity, diaphragm, and specimen position.
• Specimens should be centred before moving to higher magnification.
• Observations should be recorded accurately using descriptions, labels, drawings, and magnification.
• A microscope should be carried using two hands and handled as a precision instrument.
• Good microscope technique follows: Locate → Focus → Center → Magnify → Refocus
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