# Lesson Video: Microscopy Biology

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

16:20

### Video Transcript

In this video, we will learn how to compare and contrast different types of microscope and identify the type of microscope used by the image it produces. We will first outline the structure and features of a light microscope and learn how to calculate their total magnifying power. We will also learn what the terms magnification and resolution mean and compare these features in three different types of microscope: light microscopes, transmission electron microscopes, and scanning electron microscopes.

This diagram is of a microscope, a tool that is used in the study of microscopy and in studying all the tiny aspect of the living world. The organisms or part of an organism that we look at using a microscope are called specimens. This small yellow structure represents a single organism called an amoeba. And it is placed onto a small glass rectangular sheet called a slide. Microscopes allow us to see the distinguishing features of a small organism like this amoeba by magnifying it. Magnification is how many times larger an image appears compared with the original object. So a magnified object always appears bigger than it actually is.

Let’s look at the different parts of a light microscope, which is the specific example that we’re looking at here. Compound light microscopes usually consist of two lenses: the eyepiece lens and the objective lens. When we look through a microscope, we look through a structure called the eyepiece. The eyepiece contains the eyepiece lens, which is the lens closest to the observer’s eye. The objective lens is the one closest to the object being observed.

Light microscopes such as this one tend to have multiple objective lenses that can alter the total magnification of the specimen. Here, there are three. In order to use a light microscope, we place a slide containing our specimen onto a wide flat area called the stage and secure it with stage clips. We then look through the eyepiece and adjust the focus if the image is blurry. To do this, we would first use the larger, coarse focus knob and then the smaller, fine focus knob. Note that if you increase the magnification, your image will probably become blurry again. So you may need to adjust the fine focus knob until you can see a crisp, clear image.

A beam of light travels from the light source through the specimen on the slide and into the objective lens that we are currently using, which, in this example, is the 10 times objective lens. From there, light travels through the eyepiece lens and into the observer’s eye, where it forms an image of the object that is larger than the object itself.

On a light microscope, the magnification of the eyepiece lens remains the same, typically at around 10 times magnification. But the magnification of the objective lens you choose to use will determine the total magnification of the image that you will see. For example, if we looked through the microscope at the amoeba specimen on this slide, we might see something that looks like this. If a specimen is seen magnified 100 times, this means that it appears to be 100 times wider and 100 times longer than it really is.

By turning the objective lenses so that the 40 times objective lens clicks into place about the specimen, we have increased the magnification of the microscope, so the amoeba will appear larger. This is what the amoeba might look like at 400 times magnification using the 40 times objective lens.

The degree to which an image produced by microscope is larger than the object itself depends on the total magnifying power of the microscope. Total magnifying power, otherwise known as total magnification, can be calculated by multiplying the magnification of the eyepiece lens, which is usually 10 times in a light microscope, by the magnification of the objective lens that is currently in use. For example, we are viewing this amoeba using the highest power objective lens, which is 40 times on this light microscope. Our total magnifying power would be 10 times, the magnification of the eyepiece lens, multiplied by 40 times, the magnification of the objective lens that we are using. So the total magnifying power of our microscope and how much the image of the object is enlarged by is, in this case, 400 times.

Let’s look at the typical features of a light microscope. Light microscopes always produce two-dimensional or 2D images. This means that the images only have a length and a width and no depth, so they appear flat. Their typical maximum magnification is around 1,500 times, so they can make objects appear 1,500 times larger than they actually are. This maximum magnification is much higher than the typical magnification of commonly available microscopes such as those used in schools, however, which are typically producing images up to 400 times the size of the actual object.

Light microscopes are unique in that the specimens, which can be living or dead, can also be stained in light microscopy, which means that they can produce high-contrast, colored images. Light microscopes are most commonly used in biology to view tissues and distinguish between individual cells and some of the larger organelles within them. For example, the nucleus is often quite visible when it’s stained in light microscopy. Light microscopes allow us to view the natural colors in a specimen, for example, the green chloroplasts in a plant cell or the purple pigments in a red onion cell.

Most cells are transparent, however, which makes them difficult to distinguish from each other. To resolve this issue, stains and dyes, like Congo red pictured here, can be applied to a specimen before it is placed under a light microscope. Stains and dyes are taken up by different degrees by different cellular components, which increases the contrast between these components, making them easier to identify. Often, multiple stains are used in a process called differential staining to distinguish between and identify a mixture of components.

Let’s look at an example in the human body to visualize this better. This is a micrograph image produced by a light microscope of some human liver cells. Micrographs are effectively photographs of a magnified structure that are produced using a microscope. These liver cells have been stained with hematoxylin and eosin stain. While hematoxylin stains the nuclei of these liver cells in a purplish blue color, eosin stains the extracellular matrix and cytoplasm of these liver cells pink, which makes the structures easier to distinguish from each other.

Let’s look at another important characteristic of microscopes next, the resolving power. The resolving power of the microscope is the degree to which they can produce a high-resolution image. The resolution of any optical device, such as a microscope, is the minimum distance apart two adjacent objects must be for them to be visually distinguishable from each other. The smaller the objects are, the harder it is to distinguish them as distinct. Therefore, when viewing tiny things under a microscope, we need a strong resolving power, as they will be a very small distance from each other.

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 towards you, from a distance you will only see one light approaching. But as this car becomes closer, one light will resolve into two distinct lights, one coming from each headlight. The lights now 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 more easily. This also allows some of the finer details of the car, such as the license plate, to become more recognizable and distinct.

One of the factors affecting resolution of an optical tool is the wavelength of the source of illumination that is used, which in light microscopes is light. The very best light microscopes can resolve images up to around 200 nanometers apart, which is a very small 0.0002 millimeters apart. And this is partly limited by the wavelength of light. You can see from this diagram that light has a fairly large wavelength, which means that these two larger pink objects are far enough apart that they can be distinguished from each other with a tool that uses light, such as a light microscope.

As these two green objects are less than 200 nanometers apart, however, they cannot be distinguished from each other with a light microscope. As electron beams have a significantly smaller wavelength than light, they have a much better resolving power. So objects which are a tiny distance apart, like the green structures, still appear separate from each other. There are two main types of microscope which use electron beams instead of light as their illumination source, transmission electron microscopes or TEMs and scanning electron microscopes or SEMs.

TEMs produce magnified images at a high resolution by transmitting a beam of electrons through a specimen. The electron beam is then focused using electromagnets to produce an image. This micrograph has been produced using a transmission electron microscope. It shows the nucleolus within the nucleus of a cell. As you can see, TEMs produce two-dimensional, flat images in black and white which are highly magnified.

Let’s look at scanning electron microscopes or SEMs next. SEMs also produce highly magnified and resolved images, but they work instead by passing a beam of electrons over the surface of a specimen, which has been covered in metal ions such as gold. The metal ions reflects the electron beams, the signals from which are collected by specific detectors that produce an image of the surface of the specimen.

Let’s look at a micrograph image that has been produced by an SEM. This micrograph, produced by an SEM, shows a highly detailed surface of some pollen grains. You can see from the micrograph that SEMs, like TEMs, produce black and white images, but SEMs produce an image with a three-dimensional view of the surface of a specimen. A 3D image has a length, a width, and a depth, so they do not appear flat, much like how a cube has three dimensions, while a square has only two: a length and a width, and so is 2D.

Let’s look at the typical features of electron microscopes so that we can compare them both to light microscopes. And to do this, we will use a table. We know that the images produced by both TEMs and light microscopes are two-dimensional, but the images produced by SEMs are three-dimensional. Both transmission and scanning electron microscopes produce black and white images, though false color can be added after the micrograph has been produced. Light microscopy can show us the true color of a specimen, or stains can be added to specimens to produce high-contrast, colored images.

The preparation of specimens for electron microscopy usually results in their death. If a living specimen was subjected to an electron beam, it would definitely die so only dead specimens are ever used for electron microscopy, while in light microscopy the specimens can be living or dead. While TEMs can produce a highly detailed image of the organelles and other structures within a cell, SEMs produce detailed images of the surface of a specimen and light microscopes are usually used to view specific cells within tissues.

Both electron microscopes have a much higher maximum magnification that they can achieve than light microscopes, the very best of which can produce images magnified up to 1,500 times. While SEMs can produce magnification between one to two million times the size of the specimen itself, TEMs can produce an image magnified up to more than 50 million times the specimen’s actual size. The resolving power of electron microscopes is better than even the most sophisticated light microscopes, which can only resolve images up to 200 nanometers apart. SEMs can produce images where objects up to 0.5 nanometers apart can still be distinguished from each other, while TEMs can impressively produce images where the objects can be a tiny 0.05 nanometers apart and still appear separate.

Let’s have a go at a practice question to see how much we’ve learned about the different types of microscope.

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? A light microscope, a scanning electron microscope, or a transmission electron microscope.

Let’s start by looking at the information that we’ve been provided with by the question and the micrograph. This image is three-dimensional, and it is showing the surface of the head of the ant. It is also in black and white. Different microscopes produce different sorts of images, and we can use this information to determine what microscope produced this particular micrograph.

We are using a table to compare the three different types of microscope. Scanning electron microscopes can be shortened to SEMs, while transmission electron microscopes can be shortened to TEMs. Light microscopes and TEMs both produce two-dimensional, flat images, while SEMs produce three-dimensional images. Light microscopy shows us the natural color of specimens, which may also be stained with dyes to produce high-contrast, colored images, while images produced by the two electron microscopes can only be black and white, though they might have false color added later.

Light microscopes have the lowest magnification of the three types. And therefore, they tend to be used to view and distinguish between cells within tissues or visualize entire small living organisms. Scanning electron microscopes have a high magnifying and resolving power, and they work by passing electron beams over the surface of a specimen, which are reflected by metal ions put on the specimen’s surface. And the resulting signals are collected by specific detectors to produce highly detailed images of the surface of a specimen.

Transmission electron microscopes typically have an even higher magnifying and resolving power, and they work by passing a beam of electrons through a specimen to produce highly detailed images of intracellular structures such as organelles.

The micrograph of the ant is in 3D. Purely based on its three-dimensional nature, both light microscopes and transmission electron microscopes can be ruled out, suggesting that the image was produced by a scanning electron microscope, which does produce 3D images. The image also shows the surface of the ant’s head. And we know that SEMs typically produce images of the surface of specimens. The micrograph is not of the cells themselves nor is it of intracellular structures such as organelles, so this suggests that neither light nor transmission electron microscopes would produce this micrograph. We can therefore deduce that this image has been produced by a scanning electron microscope.

Here are some of the key points that we have covered in this video.