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.
