Exam 3 Flashcards
Name that Acronym
FCS
Fluorescence Correlation Spectroscopy
Name that Acronym
FRET
Fluorescence Resonance Energy Transfer
What is FFTs?
- Fluorescence Fluctuation Techniques
- techniques that use a confocal microscopy to measure changes in fluorescent signal and extract information from those fluctuations
What are the Three FRET Approaches?
- donor and acceptor molecules differ and the acceptor is fluorescent
- donor and acceptor molecules differ but the acceptor is not fluorescent
- donor and acceptor are the same type of molecule and acceptor fluoreces
Define Oligomer
- a polymer molecule consisting of a small number of monomers
What are the two main categories for FFTs?
- Spot Measurement
- FCS
- PCH
- Scanning Measurements
- RICS
- N&B
What is Super-Resolution Microscopy?
- obtaining image information beyond the abbe diffraction limit (about 200nm)
- resolving structures below appox. 200nm
What is SIM?
- 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
Name that Acronym
FCCS
Fluorescence Cross Correlation Spectroscopy
What is PCH?
- Photon Counting Histogram Analysis
- used to measure the molecular brightness of molecules
- when calibrated provide oligomeric information
- used to measure the molecular brightness of molecules
True or False
Both FCS and PCH are few/ single molecule techniques that are more invasive compared to FRAP
False
They are less invasive compared to FRAP
What is PALM?
- 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
Name that Acronym
RICS
Rastor Image Correlation Spectroscopy
Name that Acronym
FRAP
Fluorescence REcovery After Photobleaching
Name that Acronym
N&B
Number and Brightness Analysis
What is STED?
- 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
- using a second laser, madulating the fluerescense emission in the area producing a restricted emission area that is smaller than the defraction limit spot
True or False
For FCS, need to look at fluroescenece tags that are really low
True
needs to be low so that the fluctuations can be seen
Name that Acronym
SIM
Structured Illumination Microscopy
Name that Acronym
STED
Stimulated Emission Depletion
What is FCS?
- 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
What is 3D STORM?
- 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
What is STORM?
- 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
What is RICS?
- 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
What is FRET?
- 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)
What is N&B Analysis
- Number and Brightness Analysis
- similar to PCH when measuring the molecular brightness
- scanning version of PCH i.e. map accross whole cell
Define Astigmatism
- 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
What is Deadtime Effect?
- 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
Name that Acronym
PALM
Photo-Activated Localization Microscopy
Name that Acronym
PCH
Photon Counting Histogram
What is the difference between STORM and PALM?
- 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
What is Biplane PALM?
- “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
- creating two separate image planes on the same CCD camera
- creates the original image and off set image
What is Afterpulsing Effect?
- when the real event is followed by the spurious pulse thus overestimating the brightness values
- think of heart mur mur
What is FRAP?
- 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
- more of a diffusion of healthy cells into bleached section
- need to be careful about damage to cell b/c of high intensity laser
Name that Acronym
FFTs
Fluorescence Fluctuation Techniques
What are the two detector artifacts that affect brightness values?
Deadtime Effect
Afterpulsing Effect
What is FCCS?
- Fluorescence Cross Correlation Spectroscopy
- used to measure if two proteins are in the same complex
- can’t determine if the proteins directly interact
Name that Acronym
STORM
Stochastic Optical Reconstruction Microscopy
What are two methods to side step the deffraction limit resolution?
i.e. acheive super resolution
- ensemble focused light (PSF Engineering)
- STED
- GSD
- SIM
- probe-based (Photo-switchable dyes)
- PALM
- STORM
