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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.

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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.

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ref: Erickson-2003.07 tags: GFP FRET math CFP DsRed math date: 02-08-2008 16:04 gmt revision:1 [0] [head]

PMID-12829514 DsRed as a Potential FRET Partner with CFP and GFP

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ref: -0 tags: laser power concentration GFP mCherry calibration date: 02-01-2008 19:22 gmt revision:0 [head]

above, a set of curves for determining fluorescent protein concentration (GFP & mCherry) from received photon count in a two-photon microscope. Unfortunately, these depend on efficiency & power of the entire setup, so the curve is non-transferable to other microscopes.

one pass of mCherry @ 5x dilution did not seem the same as the others -- perhaps the reading light was left on?

% given a series of files, 
% calculate a quadratic to convert intensity to concentration. 
% assumed formula: 
% green intensity = background + const*[GFP]*laserpower^2
% red intensity = background + const*[RFP]*laserpower^2 +
%               const2[GFP]*laserpower^2
close all
basename = 'T002-gfp-100xdil-'; 
int_gfp100 = IntensReadfile('T002-gfp-100xdil-', 11, 2); 
int_gfp10 = IntensReadfile('T002-gfp-10xdil-', 8, 2);
int_mcherry10 = IntensReadfile('T002-mcherry-10xdil-', 7, 2);
int_mcherry5 = IntensReadfile('T002-mcherry-5xdil-', 7, 2);
int_mcherry5_2 = IntensReadfile('T002-mcherry-5xdil2-', 7, 2);
int_mcherry5_4 = IntensReadfile('T002-mcherry-5xdil4-', 6, 2);

bg_green = (int_gfp100(1) + int_gfp10(1))/2;
bg_red = (int_mcherry10(1) + int_mcherry5(1)...
    + int_mcherry5_2(1) + int_mcherry5_4(1))/4; 
powers = (0:0.1:1).^2;
int_gfp_all = [int_gfp100-bg_green, (int_gfp10-bg_green)/10]; 
pow_gfp_all = [powers(1:11), powers(1:8)]; 
green_intensity_perpower = pow_gfp_all'\int_gfp_all'
green_lab = ['green intensity = ' num2str(green_intensity_perpower) ' * power^2 + ' ...
    num2str(bg_green) ' (photons/10us) @ 8.7 ug/ml conc. gfp']; 

plot(sqrt(powers(1:11)), int_gfp100, 'o'); 
hold on
plot(sqrt(powers(1:8)), (int_gfp10-bg_green)/10+bg_green, 'or'); 
plot(sqrt(pow_gfp_all), pow_gfp_all * green_intensity_perpower + bg_green, 'gx'); 
legend('100x dilution','10x dilution','parabolic fit'); 
title('intensity of gfp vs. laser power normalized to 100x dilution')

int_mch_all = [(int_mcherry10-bg_red)/10, (int_mcherry5-bg_red)/20, ...
    (int_mcherry5_2-bg_red)/20, (int_mcherry5_4-bg_red)/20]; 
pow_mch_all = [powers(1:7), powers(1:7), powers(1:6), powers(1:7)]; 
red_intensity_perpower = pow_mch_all'\int_mch_all'
red_lab = ['red intensity = ' num2str(red_intensity_perpower) ' * power^2 + ' ...
    num2str(bg_red) ' (photons/10us) @ 8.7 ug/ml conc. mcherry']; 

plot(sqrt(powers(1:7)), (int_mcherry10-bg_red)/10+bg_red, 'o'); 
hold on
plot(sqrt(powers(1:7)), (int_mcherry5-bg_red)/20+bg_red, 'or'); 
plot(sqrt(powers(1:7)), (int_mcherry5_2-bg_red)/20+bg_red, 'ok'); 
plot(sqrt(powers(1:6)), (int_mcherry5_4-bg_red)/20+bg_red, 'om');
plot(sqrt(pow_mch_all), pow_mch_all * red_intensity_perpower + bg_red, 'gx'); 
legend('10x dilution','5x dilution','5x dilution(2)','5x dilution(4)','parabolic fit'); 
title('intensity of mcherry vs. laser power normalized to 100x dilution')

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ref: notes-0 tags: two-photon laser imaging fluorescence lifetime imaging FRET GFP RFP date: 01-21-2008 17:23 gmt revision:0 [head]