Super-sensitivity multiphoton frequency-domain fluorescence lifetime imaging microscopy

Abstract

We present a series of experiments that demonstrate a super-sensitive chemical imaging technique based on multiphoton frequency-domain fluorescence lifetime imaging microscopy (MPM-FD-FLIM) that shows a 2× improvement in imaging speed compared to the theoretical limit of conventional MPM-FD-FLIM. Additionally, this technique produces unprecedented sensitivity over a large range of fluorescence lifetimes. These results are achieved through simple modifications to data analysis in a conventional MPM-FD-FLIM microscope and are based on an analytical model describing the signal-to-noise ratio (SNR) of a MPM-FD-FLIM system [J. Opt. Soc. Am. A33, B1 (2016)]. Here we experimentally validate this model.

Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD

“…Spatially and temporally resolved measures of NAD(P)H and FAD can be performed in the frequency domain fluorescence lifetime imaging (FD-FLIM) or using an ultrafast laser and time-correlated single-photon counting (TCSPC) method (9,128). FD-FLIM is based on measuring phase shifts and changes in modulation at various frequencies using intensity-modulated excitation and analyzing these shifts using a nonlinear least squares method (31,34).…”

Generalized stepwise optical saturation enables super-resolution fluorescence lifetime imaging microscopy

“…In conventional two-photon microscopy, periodic modulation on the excitation source is generally implemented with an EOM/AOM controlled by a function generator [ 16 , 17 ]. Typically, the EOM/AOM is modulated by a sinusoidal signal I ( t ) = A [1 + m sin ωt )], where A is the average amplitude of the signal, m the modulation degree 0 < m ⩽ 1, and ω the angular frequency.…”

“…The experimental setup was an upgrade to the custom-built multiphoton FD-FLIM system we developed in [ 16 , 17 ]. Figure 3 shows a block diagram of the homodyne FD-FLIM method that was added to our previous system.…”

Deconvolution in Fluorescence Lifetime imaging microscopy (FLIM)

“…2 Fluorescence microscopy illuminates the samples to excite fluorophores within cells and collect the emitted light, whereas, in FLIM, additional measurements estimate the intrinsic property of the fluorophore as fluorescence lifetime. In FLIM measurements, the lifetime information can be extracted using either TD-FLIM 3,4 that measures the time delay between excited and emitted pulsed laser using an exponential fit function or FD-FLIM 5,6 that measures the relative change in magnitude and phase of the periodically modulated laser pulse. While FLIM has been leveraged in several biological applications, several crucial techniques from fluorescence microscopy has not been translated to FLIM.…”