Microscopy

Question 1

Light Microscope

Answer 1

Visible Light (400 nm - 700 nm)

Question 2

Compound microscope

Answer 2

Ocular lens (10x), objective lens (10x, 40x, 100x)
Magnification - ocular x objective
Resolution - smallest distance between two points
R = 2(wavelength) / NA, numerical aperature (ability of the lens to capture light)
Absolute limit = 0.2 um

Question 3

Super resolution fluorescent microscopy

Answer 3

Label the molecules themselves and have them glow

Question 4

Staining

Answer 4

Cells are transparent so must stain for contrast, however, staining will usually kill the cell

Question 5

Positive stain

Answer 5

Negatively charged specimen with postively charged dyes
Methylene Blue
Crystal Violet
Safranin (red)

Question 6

Negative Stain

Answer 6

Negative specimen with negative dye.
You end up seeing the profile of the cell because the background is stained.
Use to look for outside layer, capsule

Question 7

Differential staining

Answer 7

Use to tell difference between gram positive and gram negative bacteria
Steps
1. Heat mount and stain with crystal violet (all are purple)
2. Use iodine as a mordant (help retain color)
3. Decolorize with ethanol
4. Stain again with safranin
Gram positive will be purple, gram negative will be pink

Question 8

Microscopy that don’t kill the cell

Answer 8

Darkfield

Phase Contrast

Question 9

Darkfield

Answer 9

Darkfield ring for a dark background

Use to see motility

Question 10

Phase Contrast

Answer 10

Light passes through slide, as it goes through specimen, there is a difference in phase of light that produces a difference in contrast

Question 11

DIC

Answer 11

Pseudo 3D look

Question 12

Confocal Micrscope

Answer 12

Look at one plane at a time and can use computer to generate a 3D image

Question 13

Electron Microscope

Answer 13

Resolution is so much better because instead of using light, you’re using electrons instead