3.2.1.3 Methods of studying cells

Microscopes, magnification, calibration, cell fractionation and ultracentrifugation

Types of microscope

• Optical microscope

• Transmission electron microscope (TEM)

• Scanning electron microscope (SEM)

How an optical microscope works

• Beams of light are condensed through a condenser lens

• Light travels through the object then objective lens to form the initial magnified image

• Passes through eyepiece lens which further magnifies the image making it clear

Properties of an optical microscope

-Poorer resolution that EM as light has a longer wavelength

-Lower magnification

+Can produce coloured images

+Can view living samples

+Cheap, portable, easily available

+Easy to prep slides

How a TEM works

• beam of electrons condensed onto the object using an electron gun

• some electrons pass through specimens while other parts of the specimen are too dense to pass creating contrast in the image

• electrons pass the magnetic objective (focus and steer charged electrons) and magnetic projector (magnify final image) before a photomicrograph is created on the screen

Properties of TEM

+Higher resolution

+Higher magnification

-Only produces black and white images

-produces 2D images (can make 3D by layering 2D, but is very time consuming)

-sample must be in a vacuum so air particles do not deflect electrons, so samples cannot be living

-expensive

-complex staining process/need special training to operate

-may contain artefacts

How an SEM works

• a beam of electrons is condensed by an electromagnet

• electrons bounce off the surface, causing them to scatter in different directions depending on the contours

• a 3D image is generated and displayed on a computer screen

Properties of an SEM

+produces a 3D image

+specimens easier to prep as they do not need to be as thin as for TEM as electrons aren’t passing through

+high magnification

+high resolving power

-can’t observe living specimens as in a vacuum

-expensive

-creates a black and white image (can be turned into colour using a computer programme)

Staining

• Creates contrast in the image, making cells and organelles more visible

• Optical microscope: often use iodine

• Electron microscope: samples coated in heavy-metal compounds e.g. gold, lead citrate

Magnification formula

Conversions: millimeter to micrometer to nanometer

1mm = 1,000μm (micrometers) 10⁻⁶ m

1μm = 1000nm (nanometers) 1 × 10⁻⁹ m

Magnification

= how many times larger an image is compared to the object

Resolution

= the minimum distance between two objects in which they can still be viewed as separate

(determined by the wavelength of light/electrons)

Eyepiece graticule

= the scale on a glass disk inside the optical microscope that can be used to measure the size of objects you’re viewing under the microscope.

• When you change the objective lens (so changes magnification) you have to CALIBRATE the eyepiece to work out the distance between each division at that magnification

Stage micrometer

= a microscope slide with a known length scale etched into it

• Used to calibrate the eyepiece

• Scale is typically 1mm long and sub divisions are 10μm apart

How to callibrate the eyepiece graticule

1. Line up stage micrometer and eyepiece graticule while looking through the eyepiece to ensure one of the largest divisions meet

2. count the number of divisions on the eyepiece graticule that fit into one division on the stage micrometer scale

3. each division on the stage micrometer is 10μm, which can be used to calculate what one division on the eyepiece graticule is at that magnification

How to measure size of cells

Now you know what one division shows in the eyepiece graticule at that magnification, you can measure a cell.

E.g. If a cell is 12 divisions long, you multiply 12 by the length one division on the eyepiece graticule shows

Cell fractionation

= when cells are broken down so organelles are free to be separated using a homogeniser (blender)

Ultracentrifugation

= a labratory technique that separates subcellular organelles based on size and density using extreme centrifugal forces

What is the purpose of cell fractionation and ultracentrifugation?

Break down cells, releasing the organelles so they can be studied individually

Process of cell fractionation and ultracentrifugation

1. Firstly, you place the tissue into a homogeniser (blender) to break down cells, freeing and separating their organelles.

2. During homogenistaion, the cells must be kept cold to reduce enzyme activity, ensuring cell components do not beak down.

3. Additionally, the solution must be isotonic, so water does not move by osmosis, potentially causing cells to shrivel or burst.

4. The solution must also be buffered to prevent pH changes which would damage the organelles and enzymes.

5. The homogenate must be filtered to remove large debris, organelles remain in the solution (filtrate).

6. The filtrate centrifuged by placing the solution into a centrifuge which rapidly spins at increasing speeds, separating organelles.

7. The size of the centrifugal force and the density of organelles determines how the filtrate separates.

8. Heaviest organelles (nuclei first) form a pellet at the bottom of the test tube first, the remaining, smaller organelles stay suspended in the supernatant above.

9. The pellet is removed and then the solution is spun at a higher speed until the next pellet forms and the process is repeated until all organelles present are separated out.

Order of separation (heaviest to lightest)

• Nuclei

• Chloroplasts

• Mitochondria

• Lysosomes

• Endoplasmic reticulum

• Ribosomes