Module 1: Cells as the basis of life - biology

Prokaryotic cells

• Prokaryotic cells are unicellular organisms that:

  • Do NOT have nucleus or any other membrane bound organelles

  • DNA floats around in the cytoplasm, is small and circular

  • Examples: archaea, bacteria

Eukaryotic cells

• Eukaryotic cells are unicellular or multicellular organisms that:

  • Have a nucleus

  • Has membrane bound organelles

    • E.g. mitochondria, endoplasmic reticulum

  • Much more complex

  • DNA is contained within the nucleus, is straight and is a relatively large amount

  • Examples: animals, plants, protists, fungi

Light microscope - How does it work?

• Most simple and commonly used

• Uses visible light and two lenses to magnify a specimen

  • Light passes through the specimen

  • Light is refracted by the objective and ocular lenses

  • Light is refracted (bent) to form a magnified image

Light microscope - Specimen preparation

• A specimen may be a whole organism, a smear of cells or a thin slice of tissue

1. Put specimen on glass microscope slide

2. Add dye to stain certain structures

3. Add a drop of fluid and a coverslip 

Cell staining technqiue

• Staining used to enhance appearance of cell structures

  • Involves adding a dye to the specimen

  • Which stains certain cell components e.g. cell wall

Light Microscopy set up

1. Place the microscope on a flat bench and adjust it to a comfortable height

2. Plug it in and turn the light on

3. Put the lowest power objective lens (4x) into position

4. Adjust the light using the diaphragm and focus carefully

   1. Lower the objective lens using the coarse focus knob

   2. Focus using the knobs to get a clear image

   3. For a higher magnification switch to a higher objective and refocus

Magnification definition

The process of enlarging the apparent size of an object

Magnification formula

Image size / Actual size

Total magnification

The total magnification equals ocular lens magnification multiplied by objective lens magnification

Magnified size

Magnified size is the diameter of the field of view divided by the number of cells that fit across it

Microscope Magnification Summary Table

Ocular lens	Objective lens	Total magnification	Diameter of F.O.V
10x	4x	40x	4.5 mm
10x	10x	100x	450 μm
10x	100x	1000x	150 μm

Confocal microscope

• An advanced light microscope

• Works by passing a highly focused laser through the specimen

• Creates a high quality image of a section of the specimen

  • Moving the laser slightly creates another image

• A computer then enhances the images and stitches the sections all together to create a 3D image

Fluorescent microscope

• An type of light microscope

• The sample is labelled with a fluorescent substance that will attach to the structures that the scientist wants to observe. 

• The sample is illuminated with a high intensity source of light that causes the fluorescent substance to emit light. 

• This fluorescent light is directed through filters that separate it from surrounding light and the viewer is able to see only those areas of the sample that are fluorescing.

Parts of light microscope

Electron microscope - How does it work?

• Uses a beam of electrons and electromagnets to make the specimen look bigger

  • Electrons are small and sensitive and will bounce off everything

• The internal chamber of the electron microscope is under vacuum conditions (no air)

1. An electron gum shoots a beam of electrons at the specimen (in a vacuum)

   1. The electron beam is controlled by electromagnets

2. When the electrons hit/interact with the specimen, the beam gets scattered

   1. The way the electrons scatter depends on the structure of the specimen

3. This scattering is detected by different machinery and a computer turns the information into an image (called an electron micrograph)

Transmission electron microscope - How does it work?

1. Treat specimen with chemicals which give it increased structural strength as the electron beam is extremely hot

2. Water is removed from the specimen using alcohol

• As water evaporates immediately in a vacuum - which could destroy the specimen

3. Specimen is embedded in a resin

4. Cut specimen into super thin slices

5. Then one broad laser beam is shot at specimen

6. The electron micrograph is a detailed image of the inside of the specimen

• A TEM creates a 2D image of the ultrathin slice. 

• It can magnify up to 1,500,000x and has a resolution of about 2nm.

Scanning electron microscope - How does it work?

1. Treat specimen with chemicals which give it increased structural strength as the electron beam is extremely hot

2. Water is removed from the specimen using alcohol

• As water evaporates immediately in a vacuum - which could destroy the specimen

3. Specimen is coated in a thin layer of gold

4. Electron micrograph is a detailed image of the outside of the specimen

Difference between SEM and TEM

• TEM: one broad laser beam is shot at specimen

• SEM: one super fine beam is systematically scanned across the whole specimen

  • The SEM has poorer resolution (about 10nm) than TEM but gives excellent 3D images of surfaces.

Comparison: Light microscope Vs Electron microscope

Feature	Light microscope	Electron microscope
Purpose	Make a magnified image of a specimen	Make a magnified image of a specimen
Radiation type	Light	Electrons
Magnification	<20 000 X	<10 000 000 X
Resolution	Lower (best resolution = 0.25μm)	Higher (best resolution = 0.0001μm)
Price	Lower	Higher
Preparation	Fast, cheap, low expertise	Slow, expensive, high expertise
Specimen: alive?	Yes	No  → need to use vacuum = no oxygen = death
Colour?	Yes	No (black and white)

Resolution definition

Shortest distance between 2 points on a specimen that can still be distinguished by the microscope as separate entities

•  Higher resolution = clearer image

Rules for biological drawings

• Always use pencil

• Labels should be drawn using a ruler, should never cross each other  and should not have arrowheads

• Centre drawing in middle of page

• Diagram should take up about ½ the page (about 10cm by 10cm)

• Draw using simple clear lines

  • Do NOT sketch or shade

• Only draw 2 or 3 cells

• Only draw the structures that you see

• Diagram should include title of the name of specimen which should be underlined

• Record the magnification next to the diagram

  • OR

• Requires a scale bar

Cell scaled diagrams example

Scale - mm to μm

• Cells are measured using micrometres

• 1mm = 1000μm

What is a scale bar?

• A scale bar is a capped horizontal line that represents the ratio between the drawn size of the object and the actual size of the object

Calculating scale bar

Scale = Actual size of specimen / Size of drawing

1. Determine the actual size of the specimen (usually given in the question).

2. Choose a size that you will use to draw your diagram (if not specified)

3. Draw and label image

4. Determine scale bar using formula

5. Draw scale bar onto diagram

   

Size of one cell formula

Size = Diameter of field of view / Number of cells that fit across the diameter

What are organelles?

• Organelle = membrane-bound compartments within the cell

  • Each organelle performs a different function that keeps the cell alive

  • Creates small, enclosed spaces for specific processes

  • Help keep the cell organised

Nucleus - Structure and function of organelles

• Large in size

• Spherical in shape

• Contains genetic information needed for growth, repair and proper functioning

• Contains chromosomes made of DNA

• Bound by a double membrane

• Controls the cell by directing its activities

• The nucleus has pores which control the movement of substances into and out of the nucleus.

• Contains an organelle called the nucleolus 

Nucleolus - Structure and function of organelles

• Spherical in shape

• NOT bound by a membrane

• Made of protein and ribonucleic acid (RNA)

• Ribosomes are made here

  • Which play a major role in protein synthesis