Table of Contents
What is fluorescence blinking?
Fluorescence intermittency, or blinking, is the phenomenon of random switching between ON (bright) and OFF (dark) states of the emitter under its continuous excitation.
Why do fluorophores blink?
The blinking of fluorescent proteins can be caused by photoisomerization of the chromophore within the protein core to distinct protonation states (Remington 2006).
What is blinking microscopy?
The term Blink microscopy refers to the use of single-molecule imaging to exploit the fluctuating emission of fluorophores that are capable of photoswitching from a bright to a dark state (or from one emission wavelength to another) to construct superresolution images.
What is blinking in quantum dot?
Many potential applications of quantum dots (QDs) can only be realized once the luminescence from single nanocrystals (NCs) is understood. At the single-nanocrystal level, random fluctuations in the QD photoluminescence occur, a phenomenon termed blinking.
Is photobleaching Reversible?
The frequently used eCFP, eGFP, eYFP, and Citrine are all susceptible to reversible photobleaching. This light-induced and pH-dependent phenomenon leads to the generation of a nonfluorescent species which recovers spontaneously or through illumination.
Is quenching the same as photobleaching?
The two phenomena are distinct in that quenching is often reversible whereas photobleaching is not. Most quenching processes act to reduce the excited state lifetime and the quantum yield of the affected fluorophore.
Why do fluorophores bleach?
In optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it permanently is unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules.
What is FRET analysis?
The technique of fluorescence resonance energy transfer (more commonly referred to by the acronym FRET), when applied to optical microscopy, permits determination of the approach between two molecules within several nanometers (see Figure 1), a distance sufficiently close for molecular interactions to occur.
What is the flip technique?
Fluorescence Loss in Photobleaching (FLIP) is a fluorescence microscopy technique used to examine movement of molecules inside cells and membranes. The amount of fluorescence from that region is then measured over a period of time to determine the results of the photobleaching on the cell as a whole.
What is a BRET assay?
BRET, or Bioluminescence Resonance Energy Transfer, is a cell-based assay for studying protein-protein interactions. BRET relies on Forster resonance energy transfer from a bioluminescent donor to an acceptor in the presence of a substrate. Interestingly, BRET is a naturally occurring phenomenon in sea pansy.