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.
Highlights
Nanosecond temporal resolution enables new methods for wide-field imaging like time-offlight, gated detection, and fluorescence lifetime
Gated optical intensifiers (GOIs) based on microchannel plates (MCPs) allow for sub-nanosecond gating in a single image frame, and segmented GOIs can acquire multiple frames when combined with image splitting[1]
The throughput of time-correlated single photon counting (TC-SPC) is limited by the detector’s maximum count rate, and confocal microscopy relies on high excitation intensities that can cause non-linear photodamage to biological samples[11,12]
Summary
Nanosecond temporal resolution enables new methods for wide-field imaging like time-offlight, gated detection, and fluorescence lifetime. 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. We show an all-photon wide-field imaging system based on polarizing beam-splitters (PBS) and a Pockels cell (PC) This can be used to create two temporal bins or to modulate images on any timescale—from nanoseconds to milliseconds. We use this to demonstrate efficient wide-field FLIM of a multi-labeled sample, single molecules, and a biological benchmark. We demonstrate the use of a re-imaging optical cavity as a time-tospace converter to enable n-frame ultrafast imaging when combined with a Pockels cell gate
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