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ref: -2007 tags: photobleaching GFP date: 09-10-2019 01:42 gmt revision:1 [0] [head]

PMID-17179937 Major signal increase in fluorescence microscopy through dark-state relaxation (2007)

  • 5-25x increase in fluorescence yields.
  • Idea: allow the (dark) triplet states to decay naturally by keeping inter-pulse intervals of illumination greater than 1us.
  • Works for both 1p and 2p.
  • For volume imaging via 2p, I don’t think that 1um decay time is much of an issue; revisit given fluorophores after >1ms!
  • Suggests again that transition from triplet dark state to excited higher state is a prominent or significant cause of photobleaching; also suggests that triple quenching will have limited utility in scanned or pulsed 2p systems (will have more utility in 1p systems, perhaps..)
  • Atto532 dye has low intersystem crossing to the triplet state (1%) [3,5,14] .. humm.
  • 2p total photon emission seems to flatten above 100GW/cm^2 intensity.
  • 2p absorption is easily saturated independent of pulse width: for short pulses, high intensity leads to absorption to T1 state, which has high cross-section to the Tn>1 state; longer pulses give more time for single-photon absorption.
  • τp by m = 200 and hence the pulse energy by 14-fold does not have a considerable effect on G2p. This obviously indicates that the saturation of the S0 → S1 or of the T1 → Tn > 1 excitation eliminates any dependence on pulse peak intensity or energy.

{1469}
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ref: -2016 tags: fluorescent proteins photobleaching quantum yield piston GFP date: 06-19-2019 14:33 gmt revision:0 [head]

PMID-27240257 Quantitative assessment of fluorescent proteins.

  • Cranfill PJ1,2, Sell BR1, Baird MA1, Allen JR1, Lavagnino Z2,3, de Gruiter HM4, Kremers GJ4, Davidson MW1, Ustione A2,3, Piston DW
  • Model bleaching as log(F)=αlog(P)+clog(F) = -\alpha log(P) + c or k bleach=bI αk_{bleach} = b I^{\alpha} where F is the fluorescence intensity, P is the illumination power, and b and c are constants.
    • Most fluorescent proteins have α\alpha > 1, which means superlinear photobleaching -- more power, bleaches faster.
  • Catalog the degree to which each protein tends to form aggregates by tagging to the ER and measuring ER morphology. Fairly thorough -- 10k cells each FP.