Microscopy

Question 1

Pros/Cons of light microscopy

Answer 1

Pros: can see live cells with specific structures tagged
Cons: Resolution is only 0.2 um/200nm

Question 2

Define transmitted light microscopy

Answer 2

light seen is that that passes through sample

Sample sits between light and viewfinder

Question 3

Define Emitted Light microscopy

Answer 3

Molecules are excited by light and release own photons therefore the light that is seen comes from the sample themselves

Question 4

Define fluorescence microscopy

Answer 4

Fluorescent dyes (fluorochromes) stain component and absorb photons to release photons of longer wavelengths.
Background is dark
Type of emitted light microscopy

Question 5

Define confocal lasar scanning microscopy

Answer 5

type of fluorescent microscopy
Laser scans sample to remove out of focus light
3D images

Question 6

What are the three types of sample prep for fluorescence microscopy

Answer 6

1. Direct staining
2. Indirect staining
3. GFP

Question 7

Define direct staining

Answer 7

Staining used in fluorescence microscopy

Fluorochrome binds to specific component

Question 8

Define indirect staining

Answer 8

Fluorochrome directly linked to antibodies

Question 9

What are the two separate procedures in indirect staining

Answer 9

1. Primary antibody applied – made from 1 mammal, specific to antigen
2. secondary antibody applied – conjugated to fluorochrome and made in different mammal that primary

Question 10

What are some controls used

Answer 10

1. omit primary -> secondary should not bind to sample. Shows that fluorescence is specific to antigen
2. Use a pre-immune serum that lacks specific primary binding. Still use secondary. Shows that nothing else in the primary is contributing to fluorescence

Question 11

What is GFP

Answer 11

Green fluorescence protein found in jellyfish.

Used to stain live cells

Question 12

Define electron microscopy

Answer 12

Electron beam is directed to sample
Up to 0.1nm
Done in vacuum

Question 13

Define transmission electron microscopy

Answer 13

analogous to bright-field but greater resolution
Can see single cell in greater resolution
Use heavy metals to stain
Pros: Resolution for organelles
Cons: Sample must withstand electron bombardment and vacuum therefore cannot use living material
Sample must be thin enough for electrons to pass through

Question 14

Define Scanning electron microscopy

Answer 14

Specimen is dried and coated with gold/heavy metal
Pros: shows surface in 3D
Cons: requires vacuum

Question 15

Define resolution and its calculation

Answer 15

Closest spacing of 2 lines that can be distinguished

d= 0.61xwavelength/numerical aperture of object