Electro-optic imaging enables efficient wide-field fluorescence lifetime microscopy

Abstract

Nanosecond temporal resolution enables new methods for wide-field imaging like time-of-flight, gated detection, and fluorescence lifetime. The optical efficiency of existing approaches, however, presents challenges for low-light applications common to fluorescence microscopy and single-molecule imaging. We demonstrate the use of Pockels cells for wide-field image gating with nanosecond temporal resolution and high photon collection efficiency. Two temporal frames are obtained by combining a Pockels cell with a pair of polarizing beam-splitters. We show multi-label fluorescence lifetime imaging microscopy (FLIM), single-molecule lifetime spectroscopy, and fast single-frame FLIM at the camera frame rate with 103–105 times higher throughput than single photon counting. Finally, we demonstrate a space-to-time image multiplexer using a re-imaging optical cavity with a tilted mirror to extend the Pockels cell technique to multiple temporal frames. These methods enable nanosecond imaging with standard optical systems and sensors, opening a new temporal dimension for wide-field low-light microscopy.

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

“…Some of the advancements in wide-field FLIM include its implementation with Nipkow disc microscopy for fast 3-D FLIM imaging, 142 wide-field coupled with single plane illumination microscopy for high-resolution 3-D FLIM, 144 TG single photon avalanche diode (SPAD) cameras for phasor-based high speed wide-field FLIM, 145 multifrequency widefield, 146,147 and image gating by pockel cells. 148 Current wide-field FLIM systems are discussed in detail by Suhling and Hirvonen et al 149…”

Experimental teaching — A tribute to Yves Couder by the example: stroboscopy and fluorescence lifetime with a fan

“…A variation of this idea has been proposed recently in the context of fluorescence lifetime imaging for super-resolution microscopy which needs a very sensitive camera for the detection which cannot demodulate the megahertz frequencies needed for the nanosecond fluorescence lifetime to be detected. A fast electro-optical modulator is placed in from of the camera to perform the demodulation [37].…”

Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates

“…In SLIDE, frame averaging instead of pixel averaging can be performed, leading to low effective pulse repetition rate per pixel which can help increase signal levels 6 (see above). The analogue detection for FLIM is furthermore compatible with existing two photon microscopes and can significantly increase FLIM imaging speeds [19][20][21]23 .…”

“…Therefore, new technologies were developed with faster acousto-optical scanners 6,9 , polygonial scanners 10 , parallelized multi-foci excitation [11][12][13] , optical scanning 8 or sparse sampling 7 , just to name a few. For FLIM, recent developments also increased imaging speeds 14 with techniques employing multifoci 15 or widefield imaging [16][17][18][19] or increased detection speed by analogue lifetime detection [20][21][22] which permitted speeds up to video-rate 23 . Enhancing imaging speeds through spectral-encoded scanning has been successfully employed for confocal microscopy 24 , high-speed brightfield imaging 25 , quantitative phase imaging 26 and more, especially by employing the time stretch technique [25][26][27] .…”