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

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

2024 June | Jongchan Park, Liang Gao
Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for molecular-specific insights through the measurement of fluorescence decay time, with applications in various fields. However, conventional FLIM methods suffer from slow acquisition rates and limited 3D imaging capabilities due to extensive scanning in both spatial and temporal domains. Recent advancements have focused on enhancing the speed and 3D imaging capabilities of FLIM. This review explores the progress made in addressing these challenges and discusses potential future directions. Key strategies for improving FLIM speed include reducing scanning dwell time by increasing photon count rates or using analog recording. Techniques such as parallel detection in time-domain FLIM and continuous measurement of fluorescence decay waveforms have been developed to achieve faster imaging. Additionally, methods like time-folded FLIM and Compressed Ultrafast Photography FLIM (CUP-FLIM) have been introduced to capture complete fluorescence decay waveforms in a single shot, enabling high-speed wide-field acquisition. For 3D FLIM, integrating structured illumination with wide-field FLIM imaging and using FLIM tomography have been effective approaches. Light-field tomographic FLIM (LIFT-FLIM) has further advanced 3D FLIM by eliminating the need for sample rotation and enabling micron-scale resolution in biological samples. Future perspectives include the integration of computational imaging, advanced detector technologies, and deep tissue imaging. Computational FLIM leverages artificial intelligence to optimize imaging performance, while advanced detector technologies like 3D-stacked CMOS SPADs enhance sensitivity and timing performance. Deep tissue imaging using longer wavelengths in the near-infrared (NIR-II) range and integrating FLIM with diffuse optical tomography (DOT) offer promising avenues for clinical applications.Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for molecular-specific insights through the measurement of fluorescence decay time, with applications in various fields. However, conventional FLIM methods suffer from slow acquisition rates and limited 3D imaging capabilities due to extensive scanning in both spatial and temporal domains. Recent advancements have focused on enhancing the speed and 3D imaging capabilities of FLIM. This review explores the progress made in addressing these challenges and discusses potential future directions. Key strategies for improving FLIM speed include reducing scanning dwell time by increasing photon count rates or using analog recording. Techniques such as parallel detection in time-domain FLIM and continuous measurement of fluorescence decay waveforms have been developed to achieve faster imaging. Additionally, methods like time-folded FLIM and Compressed Ultrafast Photography FLIM (CUP-FLIM) have been introduced to capture complete fluorescence decay waveforms in a single shot, enabling high-speed wide-field acquisition. For 3D FLIM, integrating structured illumination with wide-field FLIM imaging and using FLIM tomography have been effective approaches. Light-field tomographic FLIM (LIFT-FLIM) has further advanced 3D FLIM by eliminating the need for sample rotation and enabling micron-scale resolution in biological samples. Future perspectives include the integration of computational imaging, advanced detector technologies, and deep tissue imaging. Computational FLIM leverages artificial intelligence to optimize imaging performance, while advanced detector technologies like 3D-stacked CMOS SPADs enhance sensitivity and timing performance. Deep tissue imaging using longer wavelengths in the near-infrared (NIR-II) range and integrating FLIM with diffuse optical tomography (DOT) offer promising avenues for clinical applications.
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