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{1473}  
PMID17179937 Major signal increase in fluorescence microscopy through darkstate relaxation (2007)
 
{1469} 
ref: 2016
tags: fluorescent proteins photobleaching quantum yield piston GFP
date: 06192019 14:33 gmt
revision:0
[head]


PMID27240257 Quantitative assessment of fluorescent proteins.
 
{540}  
PMID12829514 DsRed as a Potential FRET Partner with CFP and GFP  
{539} 
ref: 0
tags: laser power concentration GFP mCherry calibration
date: 02012008 19:22 gmt
revision:0
[head]


above, a set of curves for determining fluorescent protein concentration (GFP & mCherry) from received photon count in a twophoton microscope. Unfortunately, these depend on efficiency & power of the entire setup, so the curve is nontransferable 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 cd('/var/ftp/tim_hanson/T0020130_08/solutions'); basename = 'T002gfp100xdil'; int_gfp100 = IntensReadfile('T002gfp100xdil', 11, 2); int_gfp10 = IntensReadfile('T002gfp10xdil', 8, 2); int_mcherry10 = IntensReadfile('T002mcherry10xdil', 7, 2); int_mcherry5 = IntensReadfile('T002mcherry5xdil', 7, 2); int_mcherry5_2 = IntensReadfile('T002mcherry5xdil2', 7, 2); int_mcherry5_4 = IntensReadfile('T002mcherry5xdil4', 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_gfp100bg_green, (int_gfp10bg_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']; figure plot(sqrt(powers(1:11)), int_gfp100, 'o'); hold on plot(sqrt(powers(1:8)), (int_gfp10bg_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') xlabel(green_lab); int_mch_all = [(int_mcherry10bg_red)/10, (int_mcherry5bg_red)/20, ... (int_mcherry5_2bg_red)/20, (int_mcherry5_4bg_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']; figure plot(sqrt(powers(1:7)), (int_mcherry10bg_red)/10+bg_red, 'o'); hold on plot(sqrt(powers(1:7)), (int_mcherry5bg_red)/20+bg_red, 'or'); plot(sqrt(powers(1:7)), (int_mcherry5_2bg_red)/20+bg_red, 'ok'); plot(sqrt(powers(1:6)), (int_mcherry5_4bg_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') xlabel(red_lab)  
{538}  