Exam 3

Question 1

Name that Acronym

FCS

Answer 1

Fluorescence Correlation Spectroscopy

Question 2

Name that Acronym

FRET

Answer 2

Fluorescence Resonance Energy Transfer

Question 3

What is FFTs?

Answer 3

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

Question 4

What are the Three FRET Approaches?

Answer 4

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

Question 5

Define Oligomer

Answer 5

• a polymer molecule consisting of a small number of monomers

Question 6

What are the two main categories for FFTs?

Answer 6

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

Question 7

What is Super-Resolution Microscopy?

Answer 7

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

Question 8

What is SIM?

Answer 8

• 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

Question 9

Name that Acronym

FCCS

Answer 9

Fluorescence Cross Correlation Spectroscopy

Question 10

What is PCH?

Answer 10

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

Question 11

True or False

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

Answer 11

False

They are less invasive compared to FRAP

Question 12

What is PALM?

Answer 12

• 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

Question 13

Name that Acronym

RICS

Answer 13

Rastor Image Correlation Spectroscopy

Question 14

Name that Acronym

FRAP

Answer 14

Fluorescence REcovery After Photobleaching

Question 15

Name that Acronym

N&B

Answer 15

Number and Brightness Analysis

Question 16

What is STED?

Answer 16

• 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

Question 17

True or False

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

Answer 17

True

needs to be low so that the fluctuations can be seen

Question 18

Name that Acronym

SIM

Answer 18

Structured Illumination Microscopy

Question 19

Name that Acronym

STED

Answer 19

Stimulated Emission Depletion

Question 20

What is FCS?

Answer 20

• 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

Question 21

What is 3D STORM?

Answer 21

• 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

Question 22

What is STORM?

Answer 22

• 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

Question 23

What is RICS?

Answer 23

• 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

Question 24

What is FRET?

Answer 24

• 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)

Question 25

What is N&B Analysis

Answer 25

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

Question 26

Define Astigmatism

Answer 26

* 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

Question 27

What is Deadtime Effect?

Answer 27

* 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

Question 28

Name that Acronym PALM

Answer 28

Photo-Activated Localization Microscopy

Question 29

Name that Acronym PCH

Answer 29

Photon Counting Histogram

Question 30

What is the difference between STORM and PALM?

Answer 30

* 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

Question 31

What is Biplane PALM?

Answer 31

* "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

Question 32

What is Afterpulsing Effect?

Answer 32

* when the real event is followed by the spurious pulse thus overestimating the brightness values * think of heart mur mur

Question 33

What is FRAP?

Answer 33

* 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

Question 34

Name that Acronym FFTs

Answer 34

Fluorescence Fluctuation Techniques

Question 35

What are the two detector artifacts that affect brightness values?

Answer 35

Deadtime Effect Afterpulsing Effect

Question 36

What is FCCS?

Answer 36

* Fluorescence Cross Correlation Spectroscopy * used to measure if two proteins are in the same complex * can't determine if the proteins directly interact

Question 37

Name that Acronym STORM

Answer 37

Stochastic Optical Reconstruction Microscopy

Question 38

What are two methods to side step the deffraction limit resolution? i.e. acheive super resolution

Answer 38

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