Advancements in fluorescence lifetime imaging microscopy Instrumentation: Towards high speed and 3D

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging tool offering molecular specific insights into samples through the measurement of fluorescence decay time, with promising applications in diverse research fields. However, to acquire two-dimensional lifetime images, conventional FLIM relies on extensive scanning in both the spatial and temporal domain, resulting in much slower acquisition rates compared to intensity-based approaches. This problem is further magnified in three-dimensional imaging, as it necessitates additional scanning along the depth axis. Recent advancements have aimed to enhance the speed and three-dimensional imaging capabilities of FLIM. This review explores the progress made in addressing these challenges and discusses potential directions for future developments in FLIM instrumentation.

Keywords: 3D imaging; Bioimaging; Fluorescence lifetime; High speed imaging; Microscopy.

Fig. 1.. Fluorescence lifetime imaging.

(a) FLIM records time-resolved fluorescence decay at each spatial location. (b) Frequency-domain FLIM. (c) Time-domain FLIM.

Fig. 2.

High-speed FLIM methods. (a) The photon rate (throughput) of TCSPC-mode FLIM is limited to avoid measurement errors such as pile-up effects and counting loss. (b) The photon rate can be increased by using TCSPC with parallel detector arrays [22]. (c) Fluorescence decay waveform can be directly measured by high-speed photodetectors [23]. (d) Two-photon fluorescence lifetime dynamics of apoptosis induced cells were recorded at a video rate [24]. (e) Spectro-temporal encoded illumination enables kilohertz FLIM [25] and its application in (f) imaging flow cytometry.

Fig. 3.

High-speed FLIM methods using 2D detector arrays. (a) 0.5-megapixel SPAD camera with an acquisition rate up to 1 Hz [28] (b) 3.6 megapixel wide-field FLIM image captured by using the SPAD camera with image stitching [27]. (c) Time-folded FLIM. An optical cavity generates temporally delayed and spatially sheared replicas of fluorescent decay signal [29]. (d) The signal from the CMOS sensor and intensified CCD was used to generate fluorescence lifetime image with either an inverse retrieval method or neural network [29]. (e) Compressed ultrafast photography FLIM. The fluorescence signal is spatially mapped by the DMD, temporally sheared by the streak camera, and mapped onto the CCD sensor [31]. The fluorescence lifetime image is computationally reconstructed by solving the inverse problem. (f) High-speed fluorescence lifetime imaging of neural action potentials in live cells [30]. (g) Fluorescence lifetime imaging of neuronal cytoskeleton immunolabeled with multiple fluorophores.

Fig. 4.

3D FLIM. (a) Light-sheet microscopy with a TCSPC-based wide-field FLIM detector [42]. (b) 3D volumetric fluorescence lifetime image of a mixture of fluorescent beads and quantum dots [42]. (c) 3D fluorescence lifetime imaging with optical projection tomography. A series of time-gated fluorescence images were acquired at various projection angles by rotating the sample [46]. (d) 3D volumetric fluorescence lifetime image of a live zebrafish [48]. (e) Light-field tomography FLIM. The 2D perspective images of a 3D sample are captured by pupil selection through a scanning mirror. The resultant images are further rotated and compressed along one spatial axis by a dove prism and cylindrical lens, respectively. (f) High-resolution depth-sectioned fluorescence lifetime images of a mouse kidney tissue section.

Fig. 5.

Future perspective. (a) Compressed sensing method to overcome the information limit in FLIM [60]. (b) In vivo FLIM with a data compression ratio of 99% [59]. (c) 3D-stacked SPAD image sensor with a 45% fill factor [63]. (d) Fluorescence lifetime imaging of Convallaria Majalis sample at a video rate using the SPAD array [63]. (e) The mouse brain vasculature imaging through skin and skull. SWIR fluorescence enables deep tissue imaging beyond the NIR window [69]. (f) Lifetime characterization of SWIR fluorophores [70].