Lecture 10: Materials characterisation - Biological

Question 1

1. List the necessary requirements for samples to be imaged in SEM and TEM and explain how you can image biological samples in these two microscopes.

Answer 1

SEM

Provides a 3D image of the surface.

1. (Not cut into thin slices as it is surface.)
2. Fixation and dehydration using Critical Point Drying.
3. Coated with a thin layer of metal. The metal coating makes samples conductive.

TEM

Provides a 2d image of inner structure.

1. Cut into thin slices to allow electrons to pass right through the sample.
2. Fixed and dehydrated using CPD.
3. Embedded into resin to cut ultrathin.
4. Treated with heavy metals to increase contrast.

Question 2

2. Explain how the negative staining method works and what it is generally used for.

Answer 2

Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible.

Question 3

3. How does the immunogold method work and what kind of information can you obtain from it?

Answer 3

is a staining technique used in mostly TEM.

This staining technique follows the same patterns of the Indirect immunofluorescence: gold particles are most often attached to secondary antibodies which are in turn attached to primary antibodies designed to bind a specific antigen or another cell component. The gold increases electron scatter to give high contrast ‘dark spots’.

Question 4

5. Describe how fluorescence microscopy is done for a biological sample, using primary and secondary antibodies.

Answer 4

1. Tag proteins.
2. Primary is by attaching the flourescence to the main antibody and the secondary is by attaching the flourescence to a secondary antibody which attaches to the first.
3. The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths

Question 5

6. What do you need to consider when selecting primary and secondary antibodies for your fluorescence microscopy measurements?

Answer 5

Secondary antibody flourescence - provides signal amplification by increasing the number of fluorophore molecules per antigen.

This protocol is more complex and time-consuming than the primary (or direct) protocol above, but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.

Question 6

7. Please describe in general terms the principles of SDS-PAGE and western blot.

Answer 6

SDS-PAGE is an electrophoresis method that allows protein separation by mass.

1. SDS binds to protein, acts as a surfactant, covers the proteins’ intrinsic charge and gives them similar charge-to-mass ratios.
2. Applied electric field
3. Proteins migrate towards the anode, each with a different speed, depending on its mass.

Western blot is used to detect specific proteins in a sample.

1. Denaturation of proteins
2. Gel electrophoresis
3. A primary antibody is added which tags a specific protein
4. Secondary antibodies are added and bind to the primary antibody.
5. The secondary antibody is visualised through staining or immunofluorescence.