Velten, A. | bioRxiv Science

bioRxiv2023

Light sheet autofluorescence lifetime imaging with a single photon avalanche diode array

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, and in vivo NAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.

bioengineering↗

bioRxiv2020

Transient Room Lighting for Ambient Light Multiphoton Microscopy

Laser scanning microscopy techniques such as confocal and multiphoton fluorescence microscopy have been widely adopted by the biological research community due to their ability to monitor intact specimens at high spatial and temporal resolution. However, they have been limited for many biomedical, clinical and industrial applications by their fundamental need to operate in near absolute darkness. We present a lighting system that allows the use of light-sensitive imaging techniques in a fully-lit room by interleaving capture and illumination at a high frequency and exploiting the light averaging properties of the human eye. We use this system with a multiphoton fluorescence microscope to illustrate that this method is capable of image capture in a well-lit room on par with capture in absolute darkness. This comparison is quantified through noise analysis of the images. This system has been implemented for laser scanning microscopy but has potential for widefield fluorescence imaging suitable for open-field surgery.

bioengineering↗