Time‐gated fluorescence lifetime imaging and microvolume spectroscopy using two‐photon excitation

Abstract

SummaryA scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the twophoton exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced.The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca 2þ , or pO 2 and pCO 2 . In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting.The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations.Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2·15 ns, 2·56 ns and 3·34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics.Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 mm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.

Fluorescence lifetime imaging in biosciences: technologies and applications

“…FLIM in time-domain is primarily more advantageous than FLIM in frequency-domain because it provides a direct measurement of the fluorescence decay and, thus, a direct access to the parameter/parameters of interest 9,10,12,[40][41][42][43][44][45] . Considering the fact that many endogenous chromophores (quenched donors in FRET, NADH, NADPH) are characterised by fluorescence lifetimes in ps-range, the time-accuracy provided by FD FLIM is often insufficient.…”

“…single-and multi-gate imaging by means of either slow but highly efficient point-detectors 12,43,45 or by means of fast but low-efficiency image intensifiers 11,41,49 , are alternative TD FLIM techniques compatible with two-photon microscopy. Thereby, one or more time-gates are synchronised with the pulse train of a high-repetition rate pulsed laser, e.g.…”

Fast nonlinear spectral microscopy of in vivo human skin

“…Lifetime imaging methods typically utilize time correlated single photon counting [4] or time gating [22], spectral imaging can be performed using a set of emission filters [21,23] or dispersive optics and a PMT array [17] or CCD camera [20]. The advantage of the latter approach is that it affords detailed (high resolution) spectroscopic analysis; this was for instance demonstrated in in vivo mouse skin imaging experiments [7,8].…”

Fluorescence lifetime images and correlation spectra obtained by multidimensional time‐correlated single photon counting

“…In the past two decades, a number of FLIM techniques have been developed and applied to laser scanning microscopy (Becker et al, 2004a,b;Buurman et al, 1992;Carlsson and Liljeborg, 1998;Dowling et al, 1998;Krishnan et al, 2003;Lakowicz and Berndt, 1991;Schneckenburger et al, 1998;So et al, 1995;Squire et al, 2000;Syrtsma et al, 1998). The techniques are more or less successful in exploiting the depth resolution and the multiwavelength capability of a confocal or twophoton laser scanning microscope.…”