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Cell Biology > Analyzing Cells > Flashcards

Analyzing Cells Flashcards

1
Q

Transmitted Light

A

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

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2
Q

Fluorescence

A

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

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3
Q

Laser Scanning Confocal Microscopy

A

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

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4
Q

Limit of resolution

A

Limiting separation at which two objects appear distinct. Depends on

  1. wavelength of light
  2. Numerical Aperture of lens system
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5
Q

Numerical Aperture of lens system

A

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

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6
Q

Why oil immersion lenses give betteer resoluton

A

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

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7
Q

top vs bottom lense

A

top - objective lense
bottom - condensor lense

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8
Q

Bright Field

A

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

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9
Q

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

A

refractive index

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10
Q

Phase contrast

A

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

neg. - halow effect
extra brightness - obstructs view

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11
Q

DIC

A

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

neg. - shadow effect

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12
Q

H&E stain

A

acidic and basic properties

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13
Q

chemical stain

A

specificaly bind to specific structure

neg- introduce artifacts, no longer living

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14
Q

How tissue is prepaired

A

—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)

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15
Q

Fluorescent probes

A

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)

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16
Q

Fluorescent microscope

A

beam splicer - make sure splited light

light on one side not transmited - reglexted and twards

17
Q

Antibodies labeled with fluorescent dye

A

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

need second antibody - ampldy singnl - brighter

18
Q

STEPS: INDIRECT IMMUNOFLUORESCENCE

A
  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)
19
Q

Green fluorescent protein (GFP),

A

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

20
Q

Superresolution Fluorescence

A
  • 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

21
Q

PALM

A

Photoactivated localization microscopy:

supress - turn on and off florsences

22
Q

TIRF:

A

Total internal reflection fluorescence

23
Q

Fluorescence Microscopy Pros and Cons

A

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

24
Q

Confocal Microscopy

A

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

Cell Biology (7 decks)

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