Light Microscopy

Question 1

Magnification

Answer 1

image size/true size

Question 2

Resolution

Answer 2

Min distance between distinguishable objects in image

Magnify beyond resolving power - large object w/ no extra detail

Question 3

Prepare tissue for light microscopy

Answer 3

Fixation w/ chemicals - prevent decay
Support before cutting - embed in wax block
Sectioning - cut thin samples so light can pass through
Stain - produce contrast, determines which structures can be seen

Question 4

Histochemistry

Answer 4

Stain cells w/ spec. chemical

React w/ certain structures in cell

Question 5

Immunohisto (cyto) chemistry

Answer 5

Localise spec. tissue antigens w/ labelled antibodies

Question 6

In situ hybridisation

Answer 6

Localise spec. nucleic acid sequence in sample w/ labelled probe

Question 7

Haematoxylin and eosin (staining)

Answer 7

Nuclei (H) = purple/blue (basic dye binds to basophilic nucleic acids) 
Other structures (E) = pink (acidic dye binds to acidophilic proteins - esp. cytoplasm of muscle, RBC, epithelial cells)

Question 8

2D/3D interpretation

Answer 8

Observation depends on plane of section through specimen
Cut and collect serial sections through block
Reconstruct 3D

Question 9

Light microscope

Answer 9

Visible light transmitted through section
Light from source focussed by condenser lenses
Light pass through section
Section detail magnified by objective lens
+ magnify by eye-pieces
Resolution = 0.2 m
Section from wax block
Dewax by chemicals before staining

Question 10

Fluorescence microscopy

Answer 10

Focus light onto section 
Collect fluorescence emission light 
Sample preserved (chemical/freezing) 
Sample labelled w/ fluorescent protein 
Diff. dyes emit diff. colour light
Visualise and record as LM 
Green fluorescent protein from jellyfish (Aequorea victoria)
Immunofluorescence - image antibody labelled w/ fluorescent marker

Question 11

Confocal scanning laser

Answer 11

Often use fluorescence
Thicker sample than LM
Scanned beam focus at diff. levels of section
Detector produce image
Optical slicing - diff. images at diff. planes
Observe diff. cell layers
Fluorescent in situ hybridisation (FISH)
= DNA probe onto chromosomes (imaging genes for genetic disease)