I'm new to single-molecule localization microscopy. I'm curious about why, in DNA-PAINT or PAINT techniques (for example: jacs.2c11969), people typically use very low concentrations of imagers (below 10 nM or so). Can anyone explain the reasoning behind this? what happen if we increase the concentration of imagers?
Hello Van-Thang Nguyen
DNA-PAINT suffers from a comparably high fluorescence background signal from the imager strands in solution, and this limits their maximum concentration. This is the reason why their concentration typically varies between 0.5 and 10nM.
One can alleviate this concentration constraint by reducing the detected background intensity. To this end, approaches have been designed in which fluorescence from freely diffusing imager strands is not detected, either through various implementations of FRET or photoactivation.
For instance, in FRET-PAINT, donor labeled imager strands bind to an acceptor labeled docking strand, allowing energy transfer between them. By detecting only the acceptor fluorescence, donor labeled imager strands do not contribute to background signal and their concentration can be increased to 1200nM, consequently reducing the acquisition time.
The below attached paper will be helpful.
Article Completing the canvas: advances and challenges for DNA-PAINT...
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