Notes (taken on phone, sorry about poor formatting).
FLIM lifetime imaging
ave time before photon release. 10E-9s
adv. sep. near spectra. sensitive to ph etc. independent of intensity in or out. use w/ FRETmeasure distance. can id distribution of distances
method
time corr single photon counting. tcspc. 1p and 2p or confocal.
pulse femtosec laser. buield decay time histogram. can do spectral.
frequency mode. measure phase-shift and amp shift. need ref sample. dont need high speed laser.
Wolfgang Becker
how many separations distances can we measure?
histogram per pixel to make images
can do autofluorescence detection
multi-spectral FLIM
still effected by photobleaching – dif states bleach at dif rates
Angelika Rueck -SLIM
simultaneous 16 chn detection.
detect anaeorbic respiration (common to tumors) by NADH/FAD+ ratio.
acceptor lifetime also changes, use to improve fit. lifetime not dependent on wavelength. (could have done FRET eff from 1 chn acceptor alone
can I measure density of interacting TFs by FLIM? ie are bcd molecules clustered on. DNA
Michael Boersch
what software? (looks like mlab)
atp-synthase-gfp measure membrane diffusion of single molecules (e coli tirf)
measure 2-8nm rotary separation w/ fret.
fl is diffraction lim volume. 1nM in 1 fl is 1 molecule
id rotation dir dwell length per state. freeze rotor, count number of states.
Sophie pautot
membrane protein. clustering state w/ flim
neural junction formation, use flim to get around need to tightly balance acceptor donor ratio.
ahmed hiekal
cell autofluorescence – nadh/ fad comparison
enricho gratton
nuclear flow how proteins find targets. diffusion rate local measure is homogeneous
fcs. fluctuations in intensity to get diffusion gratton 2005. single paticle tracking when many are present? single pt fcs sensitive to binding vs diffusion. can’t see barriers.
measure intensity at multiple points away from original position. barriers create lack of spatial correlation in intensity. less than expected correlation –> penetrable barrier.
egfp-nls goes between low dna to low dna and high dna to high dna with a delay. but not between low to high.