Lesson Explainer: Microscopy Biology

In this explainer, we will learn how to compare and contrast different types of microscope and identify the type of microscope used by the image it produces.

Microscopes allow us to view anything as if through a very high-powered pair of glasses, from the detailed surface structure of materials and inner structure of cells to tiny living organisms, such as bacteria, which would otherwise be invisible to the naked eye. The earliest microscopes were in fact called “flea glasses,” as they were used to study small insects. Since then, microscopes have increased in power and sophistication to make the microscopic world living around us visible.

Microscopes produce magnification, which means that if a specimen is seen magnified 100x, it appears to be 100 times wider and 100 times longer than it really is.

Definition: Magnification

Magnification is how many times larger an image appears compared to the original object.

Let’s take a look at the different parts of a light microscope, as shown in the following figure, in order to gain a better understanding of how they work.

Compound light microscopes usually consist of two lenses, which are small yet thick transparent glass sheets. These lenses focus light shining through the object you wish to view into your eye to form an image. You can see an example of a lens in the photograph below.

Original transparent lens

Figure2

The eyepiece lens is situated at the top of the microscope. It is called the “eyepiece” as it is the section closest to your eye that you look through.

Key Term: Eyepiece

The eyepiece is the part of the microscope that is looked through by the observer. It contains the eyepiece lens, which is the lens closest to the observer’s eye. It is sometimes called the ocular lens (ocular referring to the eye or vision).

Down the body of the microscope, objective lenses follow the eyepiece lens. These lenses are called “objective” as they are closest to the “object” being observed. Many microscopes have more than one objective lens, marked with different magnifications. These objective lenses can be changed depending on the desired magnification of the specimen or slide. For example, if you want to use the 40x objective lens, you can move this objective lens until it is in line with the eyepiece lens.

Key Term: Objective Lens

Objective lenses are the lenses closest to the object being viewed under the microscope.

These two lenses converge light to form a visible image that is larger than the actual object being viewed itself, the basic principle of which can be seen in the following figure. The product of passing light through these lenses is a magnified image in the eye of the observer.

Beneath the objective lenses is the stage, a wide flat area on which to place a slide with stage clips to secure it. Slides are rectangular pieces of glass onto which a specimen is placed.

Key Term: Specimen

A specimen is a whole or part of an organism that has been collected and preserved for display, analysis, or research.

To ensure that your view of the specimen is not blurry, you can use the focus knobs on the side of the microscope to make your image clearer. When you first place a slide on the lower stage and look through the eyepiece lens, the image will probably appear blurry at first. While looking through the eyepiece lens, turn the larger knob, called the coarse-focus knob, to make the image clearer. Once the image becomes clear, turn the smaller knob, or the fine-focus knob, so that the finer details of your specimen become visible.

How To: Using a Light Microscope to Observe a Slide

  1. When using a light microscope, start from the lowest possible magnification by selecting the 10x objective lens and turning it until it clicks into place above the lower stage.
  2. Place the specimen on a slide and then place the slide onto the stage, secured by stage clips.
  3. Move the slide if necessary to ensure that the specimen is directly under the objective lens.
  4. Look through the eyepiece lens as shown in the photograph.
  5. Adjust the coarse-focus knob (larger knob) as you can see in the photograph until the image becomes clearer. This knob is used for large adjustments to focus.
  6. Move the slide if necessary to ensure that the area of the specimen you wish to observe is in view.
  7. Adjust the fine-focus knob (smaller knob) until the image becomes as clear as possible. This knob is used for small adjustments to focus.
  8. Increase the magnification if necessary by selecting the next highest objective lens (usually 20x) and turning it until it clicks into place just above the slide.
  9. Repeat steps 4, 5, and 7 to get your image in focus and, if needed, step 8 to increase the magnification again.

Portrait of Smart Little Schoolgirl Looking Under the Microscope.

Key Term: Total Magnifying Power (Total Magnification)

Total magnifying power is the combined ability of the eyepiece and objective lenses of a microscope to produce an image that is larger than the object itself.

Although the magnification of the eyepiece lens remains the same, the objective lens that you choose to use determines the overall magnification of the image that you will see. To calculate the total magnification of the image that we are viewing, we multiply the magnification of the eyepiece lens (usually 10x) by that of the objective lens that we are currently using. For example, if we were viewing a specimen and using the lowest-power objective lens (10x), our total magnification would be 10 (the magnification of the eyepiece lens) multiplied by 10 (the magnification of the objective lens). So, the total magnifying power of our microscope, and how much the image of the object is enlarged, is 100x.

The following is a simple equation that can be used to calculate the total magnifying power of a light microscope.

Formula: Calculating the Total Magnification

Totalmagnicationmagnicationoftheeyepiecelensmagnicationoftheobjectivelens=×

Example 1: Defining Magnification

Which of the following best defines magnification?

  1. Magnification is calculated as the magnifying power of the objective lens divided by the magnifying power of the eyepiece.
  2. Magnification is the number of different structures that can be identified in an image.
  3. Magnification is the minimum distance apart two objects can be in order for them to appear as separate items.
  4. Magnification is how much bigger the image is than the actual object.

Answer

We need to be careful with questions asking for the “best” definition, because even though they are multiple-choice questions, they are not easy to answer, as more than one answer may seemingly be correct.

If you think about the other ways the word “magnify” is used, they always refer to making something appear bigger than it is. For example, to magnify an issue is to make it seem more serious, and a magnifying glass makes small objects look larger. This suggests that the answer is option D. However, as we are looking for the best definition, you need to make sure the others do not apply. We can approach most multiple-choice questions using the process of elimination.

Option A is (incorrectly) telling us how to calculate the total magnifying power or total magnification of a microscope but does not actually refer to the definition of magnification itself. If you spot a calculation that is incorrect, this is an easy way to eliminate an option in a multiple-choice question.

Option B is relevant to resolution, as the number of different structures identifiable in an image depends on how easily the different objects can be resolved or be distinguished from each other. Although magnifying a structure can help make it discernible from other objects, this statement misses the key point of magnification, which is that it makes objects appear larger than they actually are.

By recalling the definition of magnification as not being the degree to which structures can be distinguished from each other, we can eliminate option C.

Our correct answer is, therefore, option D. Magnification is how much bigger the image is than the actual object.

Light microscopes are unique in that specimens, which can be living or dead, can be stained to produce color images. These microscopes are often used in biology to view tissues and distinguish between individual cells, as well as some of the larger organelles within them, such as the nucleus. They always produce two-dimensional (2D) flat images.

To more easily distinguish between cells or structures within a cell or specimen, stains and dyes can be applied to the specimen before it is placed under the light microscope. Stains and dyes are taken up with different degrees by different cellular components. This increases the contrast between the different components and makes them easier to identify.

For example, methylene blue binds to negatively charged cellular components in the cytoplasm or nucleus. As another example, Congo red is repelled by the negatively charged cytoplasm and so mostly leaves cells unstained, and instead it stains the extracellular structures surrounding the cells, making the cells easier to distinguish. Congo red can also stain cell walls of organisms like plants and fungi, or the membranes of certain bacterial cells. Multiple stains are often used to distinguish and identify a mixture of components, such as different species of bacteria. This process is called differential staining.

The following two images are examples of micrographs showing how staining can increase the visibility of certain cellular components. Micrographs are photographs of a magnified structure produced using a microscope. These micrographs were produced using a light microscope.

Light micrograph of human brain tissue showing neurons and glial cells

Figure4

The image above is a micrograph produced using a light microscope of human brain tissue showing neurons and glial cells. It shows the Nissl staining technique along with toluidine blue stain, which are often used for nervous tissue samples because they stain nucleic acids within the nuclei of the cells.

Liver cells (hepatocytes) seen with the light microscope. Their nuclei show a very large nucleolus stained in red.

Figure5

The image above is a micrograph produced by a light microscope of liver cells (hepatocytes). In this sample, hematoxylin and eosin (H&E) stain was used, where the hematoxylin stains the nuclei with a purplish-blue color and the eosin stains the extracellular matrix and cytoplasm with a pink color.

Resolution is the minimum distance apart two objects must be to be able to see them as separate. The smaller the objects are, the harder it is to distinguish them as distinct from each other. Therefore, when viewing tiny objects under a microscope, we need a high resolving power, as they will be a very small distance apart from each other.

Key Term: Resolution

The resolution of an optical device is the minimum distance between two adjacent objects at which both are visually distinguishable.

To understand resolution on a microscopic scale, let’s think about it on a large scale first. At night, if there is a car coming toward you, from a distance, you will only see one light approaching. However, as the car comes closer, that one light will resolve into two distinct lights coming from each headlight. As the car gets even closer, the lights appear further apart from each other than they did from a distance, and so the resolving power of our eyes can distinguish the two sources of light from each other.

One of the factors affecting resolution is the wavelength of the source of illumination that is being used. The best light microscopes can resolve images up to around 200 nm apart (0.0002 mm), and this is partly limited by the wavelength of light (the illumination source).

Let’s take a look at Figure 6 to see how this works.

In the top image in Figure 6, you can see that light has a fairly large wavelength and that these two objects cannot be distinguished from each other. They are too close to each other and, hence, do not meet the requirements for the minimum distance apart two objects must be for the wavelength of this illumination source.

The bottom image in Figure 6 shows an illumination source with a smaller wavelength, for example, a microscope using electrons instead of light. This results in a higher and, therefore, better resolution, allowing the same two objects to be distinguished from each other. The larger the wavelength of light, the worse (lower) the resolution.

While light has a large wavelength, and therefore tools such as microscopes that use it have a low resolution, usually around 200 nm, some light microscopes have special lenses that can resolve objects less than 100 nm apart. Electrons have a significantly smaller wavelength than that of light and so have a much better resolving power, allowing objects that are a tiny distance apart to still appear separate.

There are two main types of electron microscopes: a transmission electron microscope (TEM) and a scanning electron microscope (SEM).

Example 2: Defining Resolution

Which of the following best defines resolution?

  1. Resolution is the number of different structures that can be identified in an image.
  2. Resolution is calculated as the magnifying power of the objective lens divided by the magnifying power of the eyepiece.
  3. Resolution is how many times bigger the image is compared to the actual object.
  4. Resolution is the minimum distance apart that two objects can be in order for them to appear as separate items.

Answer

We need to be careful with questions asking for the “best” definition, because even though they are multiple-choice questions, they are not easy to answer, as more than one answer may seemingly be correct. We can approach most multiple-choice questions using the process of elimination.

Although option A is technically correct by stating that more structures can be identified when the resolution is better, it does not mention the two key points that we must consider when using the word “resolution”: it is effectively a measure of distance and it hinges on the objects appearing separate, or distinct, from each other. To help you remember this, always think about the units of a term if it has any. The unit of resolution is usually nanometres (nm), a measure of distance, and therefore our answer should refer to the distance two objects are from each other.

Option B is (incorrectly) telling us how to calculate the total magnifying power or total magnification of a microscope. So this one is referring to magnification and not resolution and can, therefore, be removed from our choices too. If you spot a calculation that is incorrect, this is an easy way to eliminate an option in a multiple-choice question.

By recalling our definition of magnification, that is, how many times larger an image is than the actual object, we can eliminate option C.

This makes option D our best answer, as it discusses the minimum distance apart that the structures must be to appear separate.

Therefore, the correct answer is that resolution is the minimum distance apart two objects can be in order for them to appear as separate items.

Both types of electron microscopes produce black-and-white images, although false color can be added after the micrograph is produced. They both have a much higher magnification than that of light microscopes (TEM: more than 50000000x; SEM: 10000002000000x). Their resolving power is better too, allowing them to distinguish far smaller objects as being separate from each other with a typical maximum resolution of 0.5 nm for SEM and 0.05 nm for TEM. While TEMs produce 2D images, often used to view organelles inside a cell in high detail, SEMs produce detailed 3D images of the surface of a specimen.

The preparation of specimens for electron microscopy usually results in their death. If a living specimen is subjected to an electron beam, it will definitely die, so only dead specimens are used for electron microscopy.

Key Term: Transmission Electron Microscope (TEM)

A transmission electron microscope transmits a beam of electrons through a specimen, which is focused using electromagnets to produce a detailed black-and-white 2D image of the internal structures of a cell or specimen.

Some examples of micrographs produced by a transmission electron microscope (TEM) can be seen in the images below.

micrograph showing an oogonium with an atypical nucleolus.

Figure7

The image above is a TEM micrograph showing an immature female reproductive cell (oogonium). The nucleolus can be seen in black, and mitochondria and lipid droplets can be seen in the cytoplasm.

False colour transmission electron microscope micrograph

Figure8

The image above is a false-color TEM micrograph showing the cytoplasm (blue) and the nuclear envelope around the nucleus (gold), attached to the rough endoplasmic reticulum (red).

Key Term: Scanning Electron Microscope (SEM)

A scanning electron microscope emits a beam of electrons over the surface of a specimen that is covered in metal ions (e.g., gold). These ions reflect the electrons, which are then collected to produce a highly detailed black-and-white 3D image of the specimen’s surface.

Let’s look at some examples of micrographs produced by a scanning electron microscope (SEM).

Varroa destructor bee parasite - an electron scanning microscope photo - Magnification 55x

Figure9

The image above is an SEM micrograph of a Varroa destructor bee parasite, magnified 55x.

Ebola electronical microscope Ebola virus under a electronical microscope

Figure10

The image above is an SEM micrograph of Ebola virus.

Example 3: Identifying Micrographs Produced by Different Microscopes

The micrograph provided is of the head of an ant. Which type of microscope is most likely to have been used to produce this image?

scanned electronic microscope ant
  1. Light microscope
  2. Transmission electron microscope
  3. Scanning electron microscope

Answer

This image is 3D, and it shows the exterior surface of a highly magnified part of an organism that appears to be dead.

Using the fact that this image is 3D, both light microscopes and transmission electron microscopes can be ruled out, suggesting that the image was produced by a scanning electron microscope. Not only does a scanning electron microscope produce micrographs with a high magnification and resolution, but also the images produced are 3D and show the surface of the specimen.

As this micrograph shows a highly magnified and resolved exterior in 3D, we can conclude that this image has been produced by a scanning electron microscope.

A useful way to compare microscopes is to use a table. Let’s look at a comparison of the magnifying power, resolution, image produced, and mechanism of light between scanning electron and transmission electron microscopes in the following table.

Example 4: Comparing the Resolution, Magnification, and Type of Image Produced by Different Microscopes

Which of the following tables correctly compares light, scanning electron, and transmission electron microscopes?

  1. Types of MicroscopeTypical Maximum Resolution (nm)Typical Maximum MagnificationImage Produced
    Light 0.51000000x2D, color
    Scanning electron20050000000x3D, black and white
    Transmission electron2001500x2D, black and white
  2. Types of MicroscopeTypical Maximum Resolution (nm)Typical Maximum MagnificationImage Produced
    Light2001500x2D, color
    Scanning electron0.51000000x3D, black and white
    Transmission electron0.0550000000x2D, black and white
  3. Types of MicroscopeTypical Maximum Resolution (nm)Typical Maximum MagnificationImage Produced
    Light 2001500x2D, black and white
    Scanning electron0.11000000x3D, color
    Transmission electron0.150000000x2D, black and white
  4. Types of MicroscopeTypical Maximum Resolution (nm)Typical Maximum MagnificationImage Produced
    Scanning electron2001500x2D, color
    Transmission electron2001000000x3D, black and white
    Light 20050000000x2D, black and white

Answer

The simplest way to approach a question like this is to use the process of elimination.

First, let’s look at the type of image produced in each example.

We know that only light microscopes can produce color images, which allows us to eliminate option C as this states that scanning electron microscopes produce color images. We also know that only scanning electron microscopes can produce 3D images, which allows us to eliminate option D as this states that transmission electron microscopes produce 3D images and that scanning electron microscopes do not.

We are left with options A and B, so let’s look at the maximum resolution and magnification. Magnification alone gives us our answer and is usually the easiest to remember. Light microscopes always have the lowest magnification, 1500x, compared to electron microscopes, which have magnifications of 1000000x for SEM and 50000000x for TEM. By looking at the magnifications, option A tells us that TEMs have a lower magnification than that of light microscopes and is therefore an incorrect answer, making B our last viable option.

Let’s check our answer by looking at the maximum resolution column. Electron microscopes can resolve objects that are much smaller than those resolvable by light microscopes. This confirms that our selection of option B is correct, as A states that light microscopes can resolve comparatively smaller objects, which is incorrect.

This can be confusing, as for a microscope to have a better ability to resolve smaller objects and distinguish them from each other, the value for the maximum resolution will be lower. This is because the maximum resolution represents the smallest possible distance apart that two objects can be from each other and still be distinguished as separate, so the smaller the value, the better.

So, our correct answer is as follows.

Types of MicroscopeTypical Maximum Resolution (nm)Typical Maximum MagnificationImage Produced
Light2001500x2D, color
Scanning electron0.51000000x3D, black and white
Transmission electron0.0550000000x2D, black and white

Key Points

  • Light microscopes produce 2D color images and usually distinguish between cells within tissues.
  • Differential staining is the use of different stains and dyes to distinguish between components in light microscopy, with either living or dead specimens.
  • Electron microscopes produce black-and-white images, which are 2D for transmission electron microscopes (TEMs), usually to view organelles, and 3D for scanning electron microscopes (SEMs), to view the details of the outer surface of a specimen.
  • Light microscopes have a maximum magnification of 1500x, whereas that of SEMs is 10000002000000x and that of TEMs is more than 50000000x.
  • Light microscopes have a maximum resolving power of 200 nm, whereas that of SEMs is 0.5 nm and that of TEMs is 0.05 nm.

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