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Fluorescence Lifetime Imaging (FLIM)

Fluorescence Lifetimes

It’s not some­thing we nor­mal­ly con­sid­er, but flu­o­res­cence takes time—once a flu­o­rophore absorbs a pho­ton, it holds on to the ener­gy for a while before emit­ting it. If you shine a very short pulse of light on a flu­o­res­cent sam­ple, the inten­si­ty of the emit­ted light decays expo­nen­tial­ly with time, and the time con­stant for this decay is called the Flu­o­res­cence Life­time.   The cool thing about the flu­o­res­cence life­time is that you can use it to inves­ti­gate the envi­ron­ment of the flu­o­rophore.

Fluorescence Quenching and FRET

Many flu­o­rophores respond to their envi­ron­ment.  E.g, in Flu­o­res­cence Res­o­nance Ener­gy Trans­fer (FRET) you excite one flu­o­rophore (the donor) which, rather than emit­ting the light, gives its ener­gy to an accep­tor flu­o­rophore if it is near­by, which emits instead.  Because the flu­o­rophores need to be close togeth­er, it allows us to mea­sure the prox­im­i­ty of mol­e­cules e.g. pro­teins in cells.  Oth­er sub­stances in the sam­ple can also act as accep­tors, and if they are not flu­o­rophores we call it quench­ing.

With­out putting in an accep­tor flu­o­rophore, just look­ing at the inten­si­ty doesn’t tell us about the quenching—a decrease in inten­si­ty could sim­ply be pho­to­bleach­ing or decreased amount of flu­o­rophore.  The great thing about the flu­o­res­cence life­time imag­ing is that it gives us two para­me­ters, the ampli­tude of the expo­nent (the amount of flu­o­rophore present) and the expo­nen­tial decay con­stant (which cor­re­sponds to the degree of quench­ing).

Developing a Fluorescence Lifetime Imaging (FLIM) system.

Fluorescence Lifetime Image of Vein Section

Flu­o­res­cence Life­time Image of Vein Sec­tion

My first ven­ture into microscopy involved devel­op­ing a flu­o­res­cence life­time imag­ing sys­tem.  I was in Paul French’s lab, work­ing with the ultra­fast lasers need­ed to pro­duce the very short puls­es to excite the flu­o­rophores.  We took the data with a time-gated image inten­si­fier, like an ordi­nary microchannel-plate image inten­si­fier, but with a very fast ‘shut­ter’ that allowed us to take an image of the emit­ted light at a speci­fic time just after the exci­ta­tion pulse.

I devel­oped the delay-line con­trol, imag­ing cap­ture and analy­sis sides of the project—writing code to con­trol the equip­ment that man­aged the shut­ter delay, take a series of pic­tures with dif­fer­ent delays, then fit sin­gle (or mul­ti­ple) expo­nen­tials, pixel-by-pixel to this time-series data.   To cal­cu­late the flu­o­res­cent life­time images, I iter­a­tive­ly least-squares fit each pix­el of a low-res scan of the image, then used this low-res image to seed the start val­ues  for iter­a­tive­ly fit­ting near­by pix­els in the high-res image.  This seed­ing method reduces the num­ber of iter­a­tions and makes the code very quick—I even man­aged to get it work­ing in real-time (which back then was quite a chal­lenge)!

An Extra Dimension of Information

This means that we can see an extra dimen­sion of information—the flu­o­res­cence lifetime—in our micro­scope images.  Flu­o­rophores now not only have a sig­na­ture absorp­tion and emis­sion spec­trum, but a char­ac­ter­is­tic life­time, which, depend­ing on the flu­o­rophore cho­sen, can change in respon­se to the envi­ron­ment, giv­ing us a extra mea­sure of what’s going on our sam­ple.


High-resolution whole field flu­o­res­cence life­time imag­ing of flu­o­rophore dis­tri­b­u­tion and envi­ron­ment Dayel, MJ.; Dowl­ing, K; Hyde, S. C.; Dain­ty, J.C.; French, P.M.W.; Vour­das, P.; Lev­er, M. J.; Dymoke-Bradshaw, Ay K.; Hares, J D.; Kel­lett, P.A. Proc. SPIE Vol. 3196, pp 111–117, 1998.Optical and Imag­ing Tech­niques for Bio­mon­i­tor­ing III

Time-domain whole-field flu­o­res­cence life­time imag­ing with opti­cal sec­tion­ing. Cole MJ, Siegel J, Webb SE, Jones R, Dowl­ing K, Dayel MJ, Parsons-Karavassilis D, French PM, Lev­er MJ, Sucharov LO, Neil MA, Juskaitis R, Wilson T. Jour­nal of Microscopy. 2001 Sep;203(Pt 3):246–57.

High res­o­lu­tion time-domain flu­o­res­cence life­time imag­ing for bio­med­ical appli­ca­tions. Dowl­ing, K; Dayel, MJ; Hyde, SCW; French, PMW; Lev­er, MJ; Hares, JD; Dymoke-Bradshaw, AKL. Jour­nal of Mod­ern Optics, 1999, Vol 46, No 2, pp 199–209.

Whole-field flu­o­res­cence life­time imag­ing with picosec­ond res­o­lu­tion using ultra­fast 10-kHz solid-state ampli­fier tech­nol­o­gy. Dowl­ing K.; Dayel M.J.; Hyde S.C.W.; Dain­ty J.C.; French P.M.W.; Vour­das P.; Lev­er M.J.; Dymoke­Brad­shaw A.K.L.; Hares J.D.; Kel­lett P.A. IEEE Jour­nal of Select­ed Top­ics in Quan­tum Elec­tron­ics, 1998, Vol.4, No.2, pp 370–375

Flu­o­res­cence life­time imag­ing with picosec­ond res­o­lu­tion for bio­med­ical appli­ca­tions. Dowl­ing K.; Dayel M.J.; Lev­er M.J.; French P.M.W.; Hares J.D.; Dymoke­Brad­shaw A.K.L. Optics Let­ters, 1998, Vol.23, No.10, pp 810–812

Two-dimensional flu­o­res­cence life­time imag­ing for in-vitro and in-vivo appli­ca­tion. French, Paul M.; Dayel, MJ.; Dowl­ing, K; Hyde, S. C.; Lev­er, M. J.; Vour­das, P.; Dymoke-Bradshaw, A K.; Hares, J D. Proc. SPIE Vol. 3250, pp 150–157, 1998. Opti­cal Biop­sy II

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