Analyzing Cells

Question 1

Transmitted Light

Answer 1

Bright-field / phase contrast / differential interference contrast -DIC
Passed through sample

Question 2

Fluorescence

Answer 2

Specific localization of organelles, macromolecular structures, and molecules within cells.
add tag to protine

Question 3

Laser Scanning Confocal Microscopy

Answer 3

Similar to Bright-field and Fluorescence, but increased clarity due to removal of out-of-focus light.
pinpoint illumination

Question 4

Limit of resolution

Answer 4

Limiting separation at which two objects appear distinct. Depends on

1. wavelength of light
2. Numerical Aperture of lens system

Question 5

Numerical Aperture of lens system

Answer 5

higher NA better resolution
(NA affects light gathering ability, related to angle of cone of light (greater with greater angle) that can enter it and refractive index of medium (how light bends/scatters). Refractive index is the ratio of speed of light in vacuum to speed of light in a particular medium

Question 6

Why oil immersion lenses give betteer resoluton

Answer 6

air glass oil
1.00 1.51 1.52
because glass and oil have the same refractive index, no interference - less light scattering - more light passing through objective lense

Question 7

top vs bottom lense

Answer 7

top - objective lense
bottom - condensor lense

Question 8

Bright Field

Answer 8

: Light transmitted straight through specimen. View living cell, but poor contrast.

Question 9

Phase of light passing through a living cell is changed according to cell’s

Answer 9

refractive index

Question 10

Phase contrast

Answer 10

: Phase alterations of light transmitted through the specimen are translated into brightness changes.

neg. - halow effect
extra brightness - obstructs view

Question 11

DIC

Answer 11

(Differential Interference Contrast): Highlights edges where there is a steep change in refractive index.

neg. - shadow effect

Question 12

H&E stain

Answer 12

acidic and basic properties

Question 13

chemical stain

Answer 13

specificaly bind to specific structure

neg- introduce artifacts, no longer living

Question 14

How tissue is prepaired

Answer 14

—Fixative applied (preserve sample)

—Embedded in paraffin wax or resin (hardened to form a solid block)

—Cut into thin slices by a microtome (~.5 um for light microscopy)

Question 15

Fluorescent probes

Answer 15

real time without killing sample

folrofloresence- tag - can be exctived with obsoption of light - excited state - unstable - beack to ground (emits light - larger wavelength than emited)

Question 16

Fluorescent microscope

Answer 16

beam splicer - make sure splited light

light on one side not transmited - reglexted and twards

Question 17

Antibodies labeled with fluorescent dye

Answer 17

Used to detect specific proteins or other molecules in cells and tissues

need second antibody - ampldy singnl - brighter

Question 18

STEPS: INDIRECT IMMUNOFLUORESCENCE

Answer 18

1. Fixation: Preserve and kill cells. Maintain cellular morphology (chemical crosslinkers: formaldehyde)
2. Permeabilization: Enable antibodies to cross cell membrane (detergent disrupts cell membrane)
3. Blocking: minimize unspecific binding of the primary antibody within the cell (normal serum, proteins such as casein)
4. Incubate with primary Antibody: Primary antibody binds to protein of interest
5. Incubate with secondary Antibody (fluorescent label): Binds to primary antibody (signal amplified)
6. Stain: localization within cell
7. Mount: fix sample on slide (prevent dehydration)

Question 19

Green fluorescent protein (GFP),

Answer 19

isolated from jellyfish. Used to monitor gene expression. GFP joined to fly promoter: active only in specialized set of neurons

view living processes - genetic engenerign - endogenous production

Question 20

Superresolution Fluorescence

Answer 20

• Florsent - bulurry - differnt debths

—Overcome limit of resolution (light microscopy) to 20 nm, order of magnitude improvement!

—If specimen contains large number of adjacent fluorescent molecules, blurry image.

supress - turn on and off florsences

Question 21

PALM

Answer 21

Photoactivated localization microscopy:

supress - turn on and off florsences

Question 22

TIRF:

Answer 22

Total internal reflection fluorescence

Question 23

Fluorescence Microscopy Pros and Cons

Answer 23

Pros:
Label specific proteins within cell using fluorescently
labeled antibodies, label multiple molecules to observeerve interaction and localization.

Super resolution fluorescence microscopy-limit of resolution is 20nm (order of magnitude below classic diffraction limit to resolution)

Cons:
Fluorescence is not permanent. Photobleaching occurs as samples are viewed

Question 24

Confocal Microscopy

Answer 24

excluding out-of-focus light - illuminating part - at specidic depth

used to resolve 3D structures of numerous objects, including networks of cytoskeletal fibers in cytoplasm and arrangement of chromosomes and genes in the nucleus.

uses pinhile - only light from focoused area gets through and lazer also goes through pinhole - specific point on sample