Microscopy

Question 1

Thiomargarita namibiensis

Answer 1

Largest bacterium known

Diameter up to 750 micrometers

Question 2

Robert Hooke

Answer 2

~1665
Coined term “cell”
Looked at piece of cork under microscope

Question 3

Antony van Leeuwenhoek

Answer 3

~1670
Made his own microscopes
First to ever see living cells

Question 4

Schlieden and Schwann

Answer 4

1838-1839
Cell theory:
All cells come from preexisting cells (Virchow and Pasteur)
All organisms have one or more cells
Cells are the basic unit of life

Question 5

Resolution

Answer 5

The ability to distinguish 2 small objects as being separate

Question 6

Resolution formula

Answer 6

d=(0.5 lambda)/(n * sin theta)
d= minimal distance that can be distinguished
lambda= wavelength (400-700 nm: visible light)
n= refractive index of medium through which light is passing
theta= half of angle of cone of light that enters lens
n sin theta: numerical aperture (function of lens; comes stamped on microscope)
Optimal: small d -> large numerical aperture

Question 7

Magnification

Answer 7

Making things appear bigger than they really are

Objective lens * ocular lens= magnification

Question 8

Light microscope

Answer 8

Light source -> condenser -> illuminate specimen -> objective (magnification; 10x, 40x, 100x, etc.) -> ocular lens (eyepiece; usually 10x)

Question 9

Bright field microscopy

Answer 9

Use stain to differentiate cell structures

Poor resolution

Question 10

Phase contrast microscopy

Answer 10

Light passed through specimen is deviated by 1/4 wavelength -> passes through phase plate: deviated ray is 1/2 wavelength out of phase -> deviated and undeviated rays cancel each other out (appearance of darkness: image)
Phase ring (attached to phase plate): converts slight differences in density and refractive index into noticeable differences

Question 11

Differential interference contrast microscopy

Answer 11

Polarizer and 2 prisms
Light goes through sample and across sample
3-D effect
Beams of light come in from side: creates shadow
Prisms split beam of light before it hits sample

Question 12

Fluorescence microscopy

Answer 12

2 sets of filters:
1st filters light before it reaches specimen, passing only those wavelengths that excite the particular fluorescent dye
2nd blocks out light and passes only those wavelengths emitted when the dye fluoresces
Fluorescent dyes can be used to identify particular cell structures
Can produce results that are based off of technique rather than what is actually happening

Question 13

Confocal laser microscopy

Answer 13

Laser light enters scope -> hits specimen on motorized stage (collects in-focus slices of specimen) -> light passes through beam-splitting mirror -> pin hole aperture blocks out of focus light
Uses fluorescence

Question 14

Confocal-scanning laser microscopy

Answer 14

Depth of light collection= depth of focus
Can see different planes of vision- can tell if things are together in the same z plane
Sample that you see is just what’s in focus
See slices of sample
Skyscraper example: can see if people are on the same floor or not

Question 15

Transmission electron microscopy

Answer 15

Similar to inverted light microscope, but uses beam of electrons instead of beam of light, and magnetic coils to focus beam instead of glass lenses
Specimen is placed in vacuum and is very thinly sliced
Contrast: heavy metal stains
Improved resolution: reduced wavelength (0.005 nm)
Resolution: 2 nm- 0.5 nm

Question 16

Scanning electron microscopy

Answer 16

3-d sample: treat with electron dense chemicals
Electrons bounce off object rather than pass through
Resolution: 3-20 nm