Test 1: Microscopy Flashcards
Describe from light source what the light goes through, and its functions.
- Diaphragm: Controls amt of light going through
- Condenser lens: focuses light from source onto specimen
- Specimen
- Objective Lens: Takes light and makes it into an image.
- Ocular Lens: Focuses light on eye.
What parts of light microscope magnifies?
Objective and Ocular
What is the site in which light is “reversed”?
Specimen, between condenser and objective lens.
Resolution of light microscope verse electro microscope is…
2-0.2 µm v. 0.1µm
The resolution is based on …
based on wave-like nature of light
The formula for resolution is…
0.61(lambda) / (nsin(theta))
Formula for Resolution: lambda is…
wavelength of light
Formula for Resolution: n is…
refractive index of medium
Formula for Resolution: theta is…
1/2 angle of rays collected by objective lens.
To increase resolution, you can increase (2) or decrease (1)
Increase n or theta, or decrease lambda.
Why is decreasing lambda the best way to increase resolution?
n is air, usually, and hence stable, and theta maximum is 1 (b/c sin theta)
Advantage sof light microscopy vere disadvantages of light microscopy?
Simple and live cells, but poor contrast.
Why is the reason for con of light microscopy?
Organelles are poor contrast because organelles are mostly H2O.
What could be done to better the con of light microscopy? What nature of light is that based on?
Based on wavelike nature of light, the organelle pushes light out-of-phase and makes it slightly darker. This push can be AMPLIFIED.
Three light microscopy amplification is…
- Bright Field Microscopy
- Phase Contrast Microscopy
- Dark Field Microscopy
Bright Field Microscopy works by…
amplifying phase contrast
Phase Contrast Microscopy works by…
Increasing contrast by amplifying phase change from passing thru organelles.
The two factors at work at Phase Contrast Microscopy are… Explain.
- Phase Plate: Pushes out of phase light ever more out of phase. Amplifies darkness.
- Differential Interference Contrast: Similar to Phase contrast, and gives psuedo-3D effect by raising dark areas and lowering light areas.
Dark Field Microscopy works by…
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.
Describe setup of Fluorescence Microscopy.
- Light source passes through one wavelength at a time.
- Filter 1 filters light to pass only particular wavelength
- Bean-Splitting Mirror focuses this light onto the specimen.
- Specimen emits another wavelength of light
- Filter 2 blocks out non-emitted light.
Confocal Flourescence Microscopy is advantageous because it…
removes light pollution caused by out-of-plane light so no need to slice sample
Electron Microscopy follows laws of resolution except at lambda. How?
lambda is not use because e- asre being bombarded. Energy of e- is directly related to Voltage is inversely related to resolutions .
What is the relationship of voltage and resolution? What is the caveat?
+ Voltage, better Resolution. BUT it damages specimen.
Electron Microscopy is simliar to Light Microscope except with 3 specialized structures. What are they and their functions/
- Power System: Voltage to accelerate e- current to magnetic lens. (btw e gun and magnetic lens)
- Vacuum Pump: Removes air around specimen beause air interferes with e-
- Magnetic Lens: Control flow of e- with electromagnets.
Describe the path of Transmission EM from e- gun.
- e-gun.
- Magnetic condenser lens (~condenser lens) focues e- stream on specimen
- Specimen
- Magnetic objective lens (~objective lens) magnifies image
- Magnetic projector lens (~occular lens)
allows images to focus onto viewing surface and magnifies - Viewing surface
Describe the path of Scanning EM from e- gun.
- e- gun
- Magnetic condenser lens
- Scanning coils causes e- beam to go back and forth over sample
- Specimen
- Detector (to the side) picks up e- that bounced off of specimen
- TV Display
What is in TEM not in SEM?
Objective and Projector Lens
What is in SEM not in TEM?
Scanning Coil and Detector
Direction of e- in TEM is ___. In SEM is ___.
unidirectional; scattered
What is the con of TEM? (2)
Can only detect e- dense Substances, because will pass thru otherwise. ALSO for dead samples only.
What is the solution of con of TEM? Con of THAT?
Stin sample with heavy metals like osmonium, but resolution is lost because heavy metals have diameters bigger than resolution.
To use TEM to determine localization of specific protein, you must…
Label selectively with heavy metals by attaching antibody specific to protein of interest with the heavy metal.
What is the con of SEM? Why?
Poorer resolution. Lower Voltage because e- must scatter off not penetrate
What is a specific step used to prepare tissue for SEM? Why so special?
Dehydration without shriveling by replacing H2O with CO2 (l). And THEN increase T to evaporate CO2.
What are 3 Fluorescent Dyes? What color are they?
- DAPI (UV-> Green)
- Rhodamine (Green -> Red)
- Fluoresceine (Blue -> Green)
