Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope

Abstract

The combination of pulsed-mode excitation multifocal multiphoton microscopy with a high-repetition, time-gated intensified CCD camera enables efficient three-dimensional (3D) fluorescence lifetime imaging. With a 200-ps gate opening at 76 MHz repetition rate, fluorescence decay can be traced in a sequence of images with varying delays between pulse and gate. Fluorophore lifetimes are measured with a precision of a few picoseconds. As an application we show that, upon two-photon excitation at 800 nm, certain pollen samples feature a multiexponential fluorescence relaxation. Our results indicate that efficient four-dimensional microscopy with hundreds of nanometer spatial and tens of picoseconds temporal resolution is within reach.

High-speed FLIM data acquisition by time-correlated single-photon counting

“…Frequency domain techniques measure the phase shift between the high-frequency modulated or pulsed excitation, and the fluorescence signal at the fundamental modulation frequency or its harmonics [27][28][29][30][31][32] . Time-domain techniques record the fluorescence decay functions directly [33][34][35][36][37][38][39] . Although the frequency-domain and the time domain are equivalent, the corresponding signal recording techniques may differ considerably in efficiency, i.e.…”

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

“…Fluorescence Lifetime Imaging (FLIM) techniques can be divided into time-domain and frequency-domain techniques, 6-9 photon counting 10,11 and analog techniques, 6,[12][13][14][15] and point-scanning 7,16 and widefield imaging techniques. 6,12,15,17 It also matters whether a technique acquires the signal waveform in a few time gates 10 or in a large number of time channels 11,18 and whether this happens simultaneously 10,11,18 or sequentially. 12,15,17 Virtually all combinations are in use.…”

“…6,12,15,17 It also matters whether a technique acquires the signal waveform in a few time gates 10 or in a large number of time channels 11,18 and whether this happens simultaneously 10,11,18 or sequentially. 12,15,17 Virtually all combinations are in use. This leads to a wide variety of instrumental principles.…”

Fluorescence-lifetime imaging with a multifocal two-photon microscope

“…Time-resolved measurements can be obtained for all the foci by integration of an ultrafast optical gated intensifier in front of the CCD camera, but it has been investigated only for the microlens approach. 5 We report here our time-resolved MMM system based on a homemade beam generator associated with a CCD camera and an optical gated intensifier that permits dynamic measurement of f luorescence with a typical resolution of 25 ps, to our knowledge the first time that it has been demonstrated with this approach.…”