Programmable Array Fluorescence Lifetime Spectroscopy/microscopy Using Tcspc

Spatial light modulators (SLMs) are a family of optical devices that allow modulation of intensity and/or phase in light microscopy to control excitation and emission. There are broadly two classes of devices employed: digital micromirror arrays (DMDs) and ferroelectric liquid crystal devices. DMDs have found use in controlled light exposure microscopy (CLEM) to reduce photobleaching and phototoxicity and in so-called programmable array microscopy (PAM). The latter have variously been used for pupil function engineering for the enhancement of resolution as in, for example, stimulated emission depletion microscopy (STED). Programmable array microscopes provide patterning of illumination or detection light in a wide-field modality [1, 2]. They have proven to be a highly versatile class of microscope due to the ability to provide optical sectioning in a widefield system. Imaging spectroscopy, confocal microscopy and fluorescence lifetime imaging, albeit in the frequency domain, have all been realised in PAM systems [3]. In this work we describe the development of new instrumentation which, like the PAM systems described above, incorporates a DMD in the primary image plane of a widefield microscope to provide patterned illumination and detection. However, unlike the PAM systems we use PMT detection and pulsed-laser excitation providing a means to record fluorescence lifetime data using time-correlated single photon counting (TCPSC). Rather than being an imaging system this can be thought of as a massively parallel fluorescence lifetime spectroscopy system. Illumination and detection of many selected regions of interest, can be performed at rates which allow an essentially hardware implemented particle tracking. Intensity thresholding is used to select regions of interest which are then tracked through time, in parallel, to provide multi-point fluorescence lifetime measurements. This is demonstrated in live cells using epidermal growth factor (EGF) tagged quantum dots in the process of binding to HER1 (EGF receptor). Using this same system we also demonstrate the versatility of the system to measure both fluorescence lifetime in a burst integrated mode and parallelised fluorescence correlation spectroscopy.