Phasor Analysis with a New Widefield Photon-Counting Flim Detector

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique for distinguishing molecular species, studying molecular interactions or assembly, and observing Forster resonance energy transfer (FRET). We present a new widefield photon-counting detector which uses the principles of phasor analysis to achieve FLIM with access to dynamics across a broad range of timescales. In previous widefield photon-counting FLIM detectors, low quantum efficiency has limited sensitivity, and global count rate limitations have constrained frame rates. Our detector, called the H33D Gen II, achieves a quantum efficiency of 15% across the visible spectrum, and achieves global count rates of several MHz. Photons are collected across the entire field of view in a widefield manner, and assigned a position in the image using a cross-strip anode. Each photon is also timed with sub-nanosecond precision using analog-to-digital converters (ADCs) and a field-programmable gate array (FPGA), which removes the dead time present in Gen I from the time-to-digital converter (TDC). This stream of photon counts is then binned into frames by software to achieve very fast frame rates, limited only by the number of photons collected and the desired statistics. We use phasor analysis with custom software to process the lifetime information, resulting in very fast generation of FLIM movies. We demonstrate the range of capabilities of this new detector and the applicability of this approach to FLIM for single particle and cellular imaging. This system provides high-precision access to nanosecond scale lifetimes, while also being capable of observing a broad timescale of dynamics (millisecond scale and higher) simultaneously across an image.