Lecture 3

Question 1

fixing tissues to image sub cellular details

Answer 1

-specimens are commonly fixed with chemicals that cross-link most proteins and DNA
-this cross-links amino groups on adjacent molecules stabilising protein-protein interactions, rendering the sample stable for further procedures
-the tissue is then embedded in paraffin wax and cut into sections using a microtome before being mounted onto a slide

Question 2

what does H&E stand for

Answer 2

haematoxylin-eosin stain

Question 3

what does H&E do?

Answer 3

stains nuclei and cytoplasm to show tissues basic morphology in different colours

it is a fairly simple staining procedure

it is variable (intensity of colour depends on person staining it)

Question 4

Common use of H&E

Answer 4

no pathologist makes a diagnosis without looking at a H&E control slide (in addition to other testing)

when using an antibody to detect a specific protein through immunohistochemistry a background stain such as H&E is used to simultaneously visualise the cells where the protein is being detected

Question 5

molecular tagging

Answer 5

can reveal disease progression

Question 6

FISH

Answer 6

fluorescent in situ hybridisation
- reveals multiple copies of the fluorescent gene instead of two
this technique can be used to see abnormalities H&E stain doesn’t show

Question 7

Fluorescence microscopy

Answer 7

• can reveal the locations of specific molecules within cells by labelling with fluorescent dyes or antibodies
• fluorescent substances absorb light of one wavelength and emit light at a longer wavelength

Question 8

immunofluorescence

Answer 8

instead of having a coloured dye that is bound to an antibody this uses fluorescent dye
the antibody is specific to a target antigen or protein and the fluorescent molecule reveals where the antigen or protein is found

Question 9

fluorochromes

Answer 9

a fluorescent molecule that glows against a dark background and can be directly or indirectly associated with the cellular molecule

multiple fluorochromes may be used simultaneously

Question 10

why use fluorescence?

Answer 10

• selective labelling
  -high contrast
  -different colours (give wavelength)

Question 11

The microscopic alphabet of dimensions

Answer 11

xyzλt

Question 12

Dimensions

Answer 12

x,y 2D
xyz 3D
xyzt (4D) timepoint
xyzλt sequential stacks for two colours (5D)

Question 13

three important parameters of microscopy

Answer 13

magnification
resolution
contrast

Question 14

Magnification

Answer 14

increase in the image size of an object

Question 15

Resolution

Answer 15

power of a microscope to distinguish between two points

always depends of contrast

Question 16

contrast

Answer 16

visible differences in brightness between parts of a sample