Authors:

Ishaan Kartik Singh

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About Paper:

Fluorescent lifetime imaging microscopy (FLIM) has been shown to have great biological relevance and potential, particularly in imaging metabolic activity of cells and Forster resonance energy transfer (FRET) studies. However, it has historically required a large number of datapoints and an accordingly slow framerate - no more than 1 Hz has been demonstrated up to this point. Faster imaging speeds is a priority in FLIM research, as it would enhance biomedical applications of FLIM, such as finding tumor microenvironments via hypoxic region detection, and open up the possibility of multiplexed FLIM, namely within super-resolution microscopy. Using the recently developed ultra-precise single photon avalanche diode (SPAD) array, which offers picosecond precision and virtually 0 noise, we demonstrate a practical implementation of SPAD arrays for rapid widefield FLIM at up to an 11 Hz framerate via fitting and 1 kHz via rapid lifetime determination (RLD). Simulations were performed to determine the set of imaging parameters that balance framerate and precision given the limits imposed by the SPAD architecture. FLIM images of quantum dots were then taken with a SPAD array at those parameters to determine that 2 Hz and 11 Hz imaging speeds recover bi-exponential fits, and 1 kHz imaging speeds recover mono-exponential fits with RLD. These findings also indicate the techniques that must be better explored to fully utilize the SPAD array's capabilities, particularly Laguerre deconvolution and bi-exponential RLD. Keywords: FLIM; SPAD; Bioimaging; Fluorescence; Single Photons

Source:

Purdue University / 2024

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Co-authors:

Ishaan Kartik Singh