Test 1: Microscopy

Question 1

Describe from light source what the light goes through, and its functions.

Answer 1

1. Diaphragm: Controls amt of light going through
2. Condenser lens: focuses light from source onto specimen
3. Specimen
4. Objective Lens: Takes light and makes it into an image.
5. Ocular Lens: Focuses light on eye.

Question 2

What parts of light microscope magnifies?

Answer 2

Objective and Ocular

Question 3

What is the site in which light is “reversed”?

Answer 3

Specimen, between condenser and objective lens.

Question 4

Resolution of light microscope verse electro microscope is…

Answer 4

2-0.2 µm v. 0.1µm

Question 5

The resolution is based on …

Answer 5

based on wave-like nature of light

Question 6

The formula for resolution is…

Answer 6

0.61(lambda) / (nsin(theta))

Question 7

Formula for Resolution: lambda is…

Answer 7

wavelength of light

Question 8

Formula for Resolution: n is…

Answer 8

refractive index of medium

Question 9

Formula for Resolution: theta is…

Answer 9

1/2 angle of rays collected by objective lens.

Question 10

To increase resolution, you can increase (2) or decrease (1)

Answer 10

Increase n or theta, or decrease lambda.

Question 11

Why is decreasing lambda the best way to increase resolution?

Answer 11

n is air, usually, and hence stable, and theta maximum is 1 (b/c sin theta)

Question 12

Advantage sof light microscopy vere disadvantages of light microscopy?

Answer 12

Simple and live cells, but poor contrast.

Question 13

Why is the reason for con of light microscopy?

Answer 13

Organelles are poor contrast because organelles are mostly H2O.

Question 14

What could be done to better the con of light microscopy? What nature of light is that based on?

Answer 14

Based on wavelike nature of light, the organelle pushes light out-of-phase and makes it slightly darker. This push can be AMPLIFIED.

Question 15

Three light microscopy amplification is…

Answer 15

1. Bright Field Microscopy
2. Phase Contrast Microscopy
3. Dark Field Microscopy

Question 16

Bright Field Microscopy works by…

Answer 16

amplifying phase contrast

Question 17

Phase Contrast Microscopy works by…

Answer 17

Increasing contrast by amplifying phase change from passing thru organelles.

Question 18

The two factors at work at Phase Contrast Microscopy are… Explain.

Answer 18

1. Phase Plate: Pushes out of phase light ever more out of phase. Amplifies darkness.
2. Differential Interference Contrast: Similar to Phase contrast, and gives psuedo-3D effect by raising dark areas and lowering light areas.

Question 19

Dark Field Microscopy works by…

Answer 19

similar to bright field EXCEPT light is from the sides not bottom, so only light that bounces off organelles are seen. Organelles look bright against dark background.

Question 20

Describe setup of Fluorescence Microscopy.

Answer 20

1. Light source passes through one wavelength at a time.
2. Filter 1 filters light to pass only particular wavelength
3. Bean-Splitting Mirror focuses this light onto the specimen.
4. Specimen emits another wavelength of light
5. Filter 2 blocks out non-emitted light.

Question 21

Confocal Flourescence Microscopy is advantageous because it…

Answer 21

removes light pollution caused by out-of-plane light so no need to slice sample

Question 22

Electron Microscopy follows laws of resolution except at lambda. How?

Answer 22

lambda is not use because e- asre being bombarded. Energy of e- is directly related to Voltage is inversely related to resolutions .

Question 23

What is the relationship of voltage and resolution? What is the caveat?

Answer 23

+ Voltage, better Resolution. BUT it damages specimen.

Question 24

Electron Microscopy is simliar to Light Microscope except with 3 specialized structures. What are they and their functions/

Answer 24

1. Power System: Voltage to accelerate e- current to magnetic lens. (btw e gun and magnetic lens)
2. Vacuum Pump: Removes air around specimen beause air interferes with e-
3. Magnetic Lens: Control flow of e- with electromagnets.

Question 25

Describe the path of Transmission EM from e- gun.

Answer 25

1. e-gun.
2. Magnetic condenser lens (~condenser lens) focues e- stream on specimen
3. Specimen
4. Magnetic objective lens (~objective lens) magnifies image
5. Magnetic projector lens (~occular lens)
   allows images to focus onto viewing surface and magnifies
6. Viewing surface

Question 26

Describe the path of Scanning EM from e- gun.

Answer 26

1. e- gun
2. Magnetic condenser lens
3. Scanning coils causes e- beam to go back and forth over sample
4. Specimen
5. Detector (to the side) picks up e- that bounced off of specimen
6. TV Display

Question 27

What is in TEM not in SEM?

Answer 27

Objective and Projector Lens

Question 28

What is in SEM not in TEM?

Answer 28

Scanning Coil and Detector

Question 29

Direction of e- in TEM is ___. In SEM is ___.

Answer 29

unidirectional; scattered

Question 30

What is the con of TEM? (2)

Answer 30

Can only detect e- dense Substances, because will pass thru otherwise. ALSO for dead samples only.

Question 31

What is the solution of con of TEM? Con of THAT?

Answer 31

Stin sample with heavy metals like osmonium, but resolution is lost because heavy metals have diameters bigger than resolution.

Question 32

To use TEM to determine localization of specific protein, you must…

Answer 32

Label selectively with heavy metals by attaching antibody specific to protein of interest with the heavy metal.

Question 33

What is the con of SEM? Why?

Answer 33

Poorer resolution. Lower Voltage because e- must scatter off not penetrate

Question 34

What is a specific step used to prepare tissue for SEM? Why so special?

Answer 34

Dehydration without shriveling by replacing H2O with CO2 (l). And THEN increase T to evaporate CO2.

Question 35

What are 3 Fluorescent Dyes? What color are they?

Answer 35

1. DAPI (UV-> Green)
2. Rhodamine (Green -> Red)
3. Fluoresceine (Blue -> Green)