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Microscopy > Exam 3 > Flashcards

Exam 3 Flashcards

1
Q

Name that Acronym

FCS

A

Fluorescence Correlation Spectroscopy

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2
Q

Name that Acronym

FRET

A

Fluorescence Resonance Energy Transfer

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3
Q

What is FFTs?

A
  • Fluorescence Fluctuation Techniques
  • techniques that use a confocal microscopy to measure changes in fluorescent signal and extract information from those fluctuations
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4
Q

What are the Three FRET Approaches?

A
  1. donor and acceptor molecules differ and the acceptor is fluorescent
  2. donor and acceptor molecules differ but the acceptor is not fluorescent
  3. donor and acceptor are the same type of molecule and acceptor fluoreces
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5
Q

Define Oligomer

A
  • a polymer molecule consisting of a small number of monomers
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6
Q

What are the two main categories for FFTs?

A
  1. Spot Measurement
    1. FCS
    2. PCH
  2. Scanning Measurements
    1. RICS
    2. N&B
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7
Q

What is Super-Resolution Microscopy?

A
  • obtaining image information beyond the abbe diffraction limit (about 200nm)
    • resolving structures below appox. 200nm
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8
Q

What is SIM?

A
  • Structured Illumination Microscopy
  • addition of regularly repeating diffraction grids at an angle to obtain super resolution images
  • 2 patterns superposed multiplicatively give rise to moire fringes
    • illuminate the sample with a light pattern
    • observe moire fringes between the pattern and the sample structure
    • deduce unresolvable information about the sample
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9
Q

Name that Acronym

FCCS

A

Fluorescence Cross Correlation Spectroscopy

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10
Q

What is PCH?

A
  • Photon Counting Histogram Analysis
    • used to measure the molecular brightness of molecules
      • when calibrated provide oligomeric information
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11
Q

True or False

Both FCS and PCH are few/ single molecule techniques that are more invasive compared to FRAP

A

False

They are less invasive compared to FRAP

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12
Q

What is PALM?

A
  • Photo-activated locolization microscopy
  • a type of super-resolution microscopy
  • same concept as STORM
    • used photoswitchable fluorescent probes to resolve spatial differences in dense populations of molecules with superresolution
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13
Q

Name that Acronym

RICS

A

Rastor Image Correlation Spectroscopy

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14
Q

Name that Acronym

FRAP

A

Fluorescence REcovery After Photobleaching

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15
Q

Name that Acronym

N&B

A

Number and Brightness Analysis

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16
Q

What is STED?

A
  • Stimulated Emission Depletion
  • creates super resolution images by the selective deactivation of flurophores, minimising the area of illumination at the focal point thus enhancing the achievable resolution for a given system
    • using a second laser, madulating the fluerescense emission in the area producing a restricted emission area that is smaller than the defraction limit spot
      • depletion laser used to decrease the size of the excitation volume
    • an increase in detection intensity increases resolution improvement and vice versa
17
Q

True or False

For FCS, need to look at fluroescenece tags that are really low

A

True

needs to be low so that the fluctuations can be seen

18
Q

Name that Acronym

SIM

A

Structured Illumination Microscopy

19
Q

Name that Acronym

STED

A

Stimulated Emission Depletion

20
Q

What is FCS?

A
  • Fluorescence Correlation Spectroscopy
  • measurement of fluorescence fluctuations produced by movements of fluorescent molecules through small detection volumes
    • fluctuations are analyzed by the auto corrrelation function
21
Q

What is 3D STORM?

A
  • a cylindrical lens is inserted into the light path to create astigmatism
  • the PSF shape changes depending on the position above and below the focal plan
  • this positional information can be used to determine exact 3D position of molecules
    • use astigmatism by back calculating the spesific depth of sample
22
Q

What is STORM?

A
  • Stochastic Optical Reconstruction Miscroscopy
  • super-resolution microscopy
  • a flurescence image is constructed from high-accuracy localization of individual flurescent molecules that are switched on and off using light of different colors
    • the flurescent molecules are being turned on and off at random positions in the sample; the images are then superimposed to create one complete image
23
Q

What is RICS?

A
  • Rastor Image Correlation Spectroscopy
  • noninvasive technique to detect and quantify events in a live cell
    • laser scanning speed is critical for accurate measurements using this technique
24
Q

What is FRET?

A
  • Fluorescence Resonance Energy Transfer
  • mechanism describing energy transfer between two light -sensitive molecules
  • indicates protein interaction and close spatial proximity
  • can be used to measure distances between proteins (A)
25
Q

What is N&B Analysis

A
  • Number and Brightness Analysis
  • similar to PCH when measuring the molecular brightness
    • scanning version of PCH i.e. map accross whole cell
26
Q

Define Astigmatism

A
  • a defection in the eyes or lens caused by a deviation from shperical curvature
    • results in distored images, as light rays are prevented from meeting at a common focus
27
Q

What is Deadtime Effect?

A
  • the time after each event during which the system is not able to record another event
    • underestimatates brightness values
    • think of a camera and waiting to take another picture
28
Q

Name that Acronym

PALM

A

Photo-Activated Localization Microscopy

29
Q

Name that Acronym

PCH

A

Photon Counting Histogram

30
Q

What is the difference between STORM and PALM?

A
  • STORM
    • the photo-switchable dye is attached to the protein of interest through antibodies
  • PALM
    • use photo-switchable protein flurophores to genetically tag the protein of interest
31
Q

What is Biplane PALM?

A
  • “3D PALM”
  • uses an extra bean splitter and mirror with a CCD at the end of the schematic
    • creates the original image and off set image
      • creating two separate image planes on the same CCD camera
        • allows to see on the x y and z planes
32
Q

What is Afterpulsing Effect?

A
  • when the real event is followed by the spurious pulse thus overestimating the brightness values
    • think of heart mur mur
33
Q

What is FRAP?

A
  • Fluorescence Recovery After Photobleaching
  • measuring the time it takes for a bleached section to “revover”
    • more of a diffusion of healthy cells into bleached section
      • used to measure diffusion rates in cells
  • need to be careful about damage to cell b/c of high intensity laser
34
Q

Name that Acronym

FFTs

A

Fluorescence Fluctuation Techniques

35
Q

What are the two detector artifacts that affect brightness values?

A

Deadtime Effect

Afterpulsing Effect

36
Q

What is FCCS?

A
  • Fluorescence Cross Correlation Spectroscopy
  • used to measure if two proteins are in the same complex
  • can’t determine if the proteins directly interact
37
Q

Name that Acronym

STORM

A

Stochastic Optical Reconstruction Microscopy

38
Q

What are two methods to side step the deffraction limit resolution?

i.e. acheive super resolution

A
  • ensemble focused light (PSF Engineering)
    • STED
    • GSD
    • SIM
  • probe-based (Photo-switchable dyes)
    • PALM
    • STORM

Microscopy (2 decks)

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