Introduction

Question 1

Detection

Answer 1

Ability to detect presence of an object

Question 2

Resolution

Answer 2

Ability to discriminate between 2 points

Question 3

Resolving power (w/ limit values)

Answer 3

The closest 2 objects can be and still be separately distinguished
Eye: 250 microns
Light microscope: 0.25 microns
Electron microscope: 0.001 microns

Question 4

Numerical aperture

Answer 4

Amount of light that a microscope can gather,
Determines resolving power
(High NA —> high resolving power)

Question 5

Resolving power (equation for calculation)

Answer 5

Resolving power = 0.61x Wavelength/ NA

Question 6

Magnification, limit

Answer 6

Only useful w/ good resolution,
Max magnification depends on numerical aperture (NA) of the lens

-empty magnification = when no more resolution w/ increased mag.

Question 7

Brightfield

Answer 7

Light microscope technique, use visible light and stained specimen

Question 8

Phase contrast

Answer 8

Type of LM,
For living specimens,
2 light beams out of phase

Question 9

differential interference contrast “DIC”/Nomarski

Answer 9

LM type,
use polarize single beam into 2,
for live specimen
*emphasize amplitude contrast

Question 10

reference cell for size comparisons

Answer 10

RBC (red blood cell)

= 7 microns!

Question 11

Laser scanning confocal

Answer 11

type of LM,
used for optical sectioning
- use laser light

Question 12

condensor

Answer 12

part of LM microscope,

produces cone of light through specimen

Question 13

objective lens

Answer 13

part of LM microscope,

enlarges and projects image into eyepiece

Question 14

eyepiece

Answer 14

part of LM microscope,

magnifies image x10, projects image onto retina

Question 15

specimen prep steps

Answer 15

1. fixation (preserve bio structure)
2. dehydration/clearing
3. embedding (facillitate sectioning, ie: wax)
4. sectioning
5. Mount
6. stain (impart contrast)

Question 16

hematoxylin

Answer 16

+ charged dye, blue-ish purple.
for “basophilic” components
esp: DNA, RNA, GAGs (proteoglycans)

Question 17

eosin

Answer 17

• charge pink dye,
  for “eosinophilic” components
  ie: proteins (NH3+)

Question 18

azan

Answer 18

stains collagen blue

Question 19

iron hematoxylin

Answer 19

stains mitochondria

Question 20

silver stain

Answer 20

stains Golgi apparatus and reticular fibers

collagen type III

Question 21

toluidine blue

Answer 21

stacked cells –> purple,

stains mast cells and RNA

Question 22

Voerhoeff

Answer 22

stains elastin black

Question 23

periodic acid schiff

Answer 23

converts glycogen and GAGs to aldehyde groups,

aldehydes –> intense magenta

Question 24

enzyme histochemistry

Answer 24

to pinpoint location of specific enzymes in cell

Question 25

immunocytochemistry

Answer 25

use antibodies to pinpoint location of specific molecs

• direct: marker conugated to antibody
• Indirect: use secondary antibody to mark primary
• immunofluorescence: secondary antibody has fluorescent probe

Question 26

In situ hybridization

Answer 26

to detect location of specific mRNAs

w/ radioactive labeled probes

Question 27

TEM (transmission electron microscopy)

Answer 27

electrons pass THROUGH specimen
2D image
high resolution

Question 28

SEM (scanning electron microscope)

Answer 28

electrons scattered/emitted by specimen

3D, surface structures

Question 29

immunogold labeling

Answer 29

localize specific proteins/antigens

Question 30

negative staining

Answer 30

to visualize details of large macromolecs

ie: DNA, proteins, viruses