Methods of studying cells

In a nutshell

The development of microscopes has allowed us to observe and investigate different cells and their internal structures. Different types of microscopes, including the optical microscope and electron microscope, exist that have their own advantages and disadvantages. In this summary you will learn about the different types of microscope and their properties.

Magnification

Definition

Magnification is how much bigger an image is compared to the size of the real object. Microscopes magnify images linearly which means that the width of the image will be magnified the same as the length.

Example

If the magnification is $\times150$ , the length of the image will be $150$  times greater than the actual object and the width of the image will also be $150$  times greater than the actual object.

$Magnification = \frac{size\space of\space image}{size\space of\space real\space object}$

Example

You are using a microscope and a cell in your image is $7.5\ mm$ . The actual size of the image is $0.05\ mm$ . What is the magnification?

$\begin{aligned}Magnification &= \frac{size\space of\space image}{size\space of\space real\space object}\\\\&= \frac{7.5}{0.05}\\\\&=\underline{150}\end{aligned}$

$\underline{Therefore,\ the\ magnification\ is\ \times150}$

Resolution

Definition

The resolution of the image is how clear the details of the image are. So, the resolution is the minimum distance two objects must be for them to appear as separate objects.

Example

For a light microscope, the resolution is around $0.2\ \mu m$ . So, objects that are $0.2\ \mu m$  or more apart will be distinguished as two separate objects.

Increasing the magnification will increase the size of the image but the resolution will limit what you can observe. You may be able to make the image bigger but it will just become more blurred.

Types of microscope

The different types of microscope work in different ways. The diagram on the left below shows how an optical microscope works. On the right is how a standard scanning electron microscope works.

Biology; Cells; KS5 Year 12; Methods of studying cells — 1. Light source, 2. Electron source, 3. Condenser lens, 4. Magnetic condenser, 5. Object, 6. Objective lenses, 7. Magnetic objective, 8. Intermediate image, 9. Eyepiece lenses, 10. Magnetic projector, 11. Human eye, 12. Fluorescent screen

Optical microscopes

Optical microscopes, also known as light microscopes, use visible light to magnify images. They allow magnification up to $\times1500$ which allows you to view some internal cellular structures. However, the resolution is around $0.2\ \mu m$ . Ribosomes are around $20\ nm$ , therefore they can not be viewed using a light microscope. Mitochondria may be viewed but not easily distinguished. Despite their limitations, they are often still used because they are cheap and easy to use.

Electron microscopes

Electron microscopes use electrons to produce an image and they have a much higher resolution than optical microscopes. Their resolution is around $0.0002\ \mu m$ and their maximum magnification is around $\times1,500,000$ . The wavelength of the electrons is far smaller than the visible light spectrum. This explains why the resolution of an electron microscope is far greater than an optical microscope.

Note: A photograph of the image seen using a microscope is called a photomicrograph.

Transmission electron microscopes (TEM)

TEM are a type of electron microscope that consist of an electron gun. This electron gun produces a beam of electrons that is focused onto the specimen. The parts of the specimen that absorb the electrons appear dark and parts that do not absorb the electrons appear bright. A black and white image is formed on a screen that can be photographed to form a photomicrograph.

Advantages	Disadvantages
The resolution of the TEM is very high at $0.1\ nm$ .	Achieving this high resolution requires a high energy electron beam which can destroy the specimen.
TEM can also magnify images up to two million times which allows you to view the internal structures of organelles.	Preparing the specimen is difficult as it must be stained with metal salts.
	The specimen must be thin to allow the electron beam to pass through.
	As the specimen is thin, a $2D$ image is formed. Sections of the specimen can be taken and layered to get an insight into the whole specimen, but this is a long and complex process.
	The system must be in a vacuum which means that living specimens can’t be observed using a TEM.
	Artefacts are not uncommon. These are remnants of the preparation procedure that can be seen on the photomicrograph but are not actually part of the specimen.

Scanning electron microscopes

SEM were developed in the $1960s$ and uses electrons to view images. The SEM directs a beam of electrons to the surface of a specimen from above. The beam bounces back and forth on the surface of the specimen before being scattered in a particular pattern that represents the contours of the surface.

Advantages	Disadvantages
Computer analysis can produce a $3D$ image.	The system must be in a vacuum which means that living specimens can’t be observed using a SEM.
The image is black and white, but the computer can be used to add false colour.	SEM has a lower resolution than TEM (around $20\ nm$ ). *Note:* This is still a much higher resolution than a light microscope.
The magnification of the SEM ranges between $\times 15$ to $\times200,000$ .
The specimen does not need to be very thin as the electrons are not having to penetrate the specimen.

Laser scanning microscopes

These are also called confocal microscopes. They use lasers to scan an object and assemble pixel information to form an image. A computer is required to scan and create the images. Laser scanning microscopes have high resolution and can be used to focus on structures at different depths. This is called depth selectivity. Laser scanning microscopes are most commonly used in medicine.

Preparing a specimen

Before using an optical microscope, you must prepare a slide of your specimen.

Procedure

1.	Pipette a drop of water onto your slide.
2.	Use tweezers or forceps to transfer your specimen to the slide.
3.	Pipette a drop of the stain to the slide. Examples Eosin is used to stain the cytoplasm. Iodine is used to stain starch grains.
4.	Carefully add a cover slip on top of the slide. *Note:* Be careful not to get any air bubbles under the cover slip otherwise your view will be obstructed.

Note: This is a temporary preparation. A permanent preparation involves holding the coverslip in place with a resin.

Cell fractionation

Cell fractionation is a process used to separate organelles from the rest of the cell in order to view the organelles under an electron microscope. There are three main stages to cell fractionation as described below.

Homogenisation

Homogenisation is the process of breaking up cells. Cells can be broken by vibration or in a blender. This breaks the plasma membrane and releases the organelles. The fluid that is left is called the homogenate. It must be kept on ice to minimise the activity of enzymes that may break down the organelles.

The solution must also be isotonic and have the same water potential as the tissue to prevent damage to the organelles by osmotic gain or loss of water. The homogenate should also be buffered to maintain the pH as any change in the pH could alter the enzyme activity or the organelle structure.

Filtration

The homogenate is filtered through gauze to remove any large debris.

Ultracentrifugation

Following filtration, the solution is a mixture of organelles that must be separated before being viewed.

Biology; Cells; KS5 Year 12; Methods of studying cells — 1. Cell fragments on ice, 2. Cell fragments in a homogeniser, 3. Homogenised tissue in a centrifuge, 4. Resultant tube, A1. Supernatant, B1. Sediment, 5. Resultant tube, A2. Supernatant, B2. Sediment, 6. Resultant tube, A3. Supernatant, B3. Sediment

Procedure

1.	The cell fragments are transferred into a tube.
2.	The tube is put into a centrifuge and spun at a low speed.
3.	The heaviest organelles, including the nuclei, are pushed to the bottom of the tube. They form a pellet at the bottom of the tube whilst the rest of the organelles remain in solution. The solution is called the supernatant.
4.	The supernatant is removed and transferred to another tube.
5.	The new tube is placed in a centrifuge and spun at a higher speed.
6.	The next heaviest organelles, the mitochondria, are pushed to the bottom of the tube and form a pellet.
7.	The supernatant is removed and transferred to a new tube.
8.	The new tube is centrifuged at an even higher speed.
9.	The process is repeated at progressively higher speeds until all organelles have been separated.

Note: The order from heaviest to lightest organelles is nucleus, (chloroplasts if it’s a plant cell), mitochondria, lysosomes, endoplasmic reticulum and ribosomes.