SignificanceFluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples.AimWe aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM.ApproachA single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells.ResultsAn NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval 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 30 × to 6 × acquisition speed advantage for the light sheet compared to the laser scanning geometry.ConclusionsFLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.
Metabolic control of immune cell function has emerged as a key regulatory mechanism in immunity. Current studies rely mainly on in vitro and ex vivo approaches to characterize the intracellular metabolism of immune cells. Here, we show that fluorescence lifetime imaging microscopy of metabolic coenzymes, NAD(P)H and FAD, captures dynamic changes in the metabolic activity of macrophages during diverse inflammatory responses in their native interstitial microenvironments. This imaging-based approach also provides single-cell resolution, in contrast to bulk measurements provided by traditional biochemical assays, making it a potentially valuable tool in the field of immunometabolism.
Breast cancer is the second most common cancer among women in the United States. Heterogeneity in breast cancer treatment response across patients indicates that patient-specific treatment screens will reduce under-treatment of aggressive and recurrent cancers, while also sparing patients with non-aggressive disease toxicities due to overtreatment. Xenografts grown from patient-specific tissue in zebrafish present a novel platform for a medium to high-throughput anti-cancer drug screen to individualize patient therapy. The goal of this project is to develop an optical imaging anti-cancer drug screen to evaluate patient-specific zebrafish tumors. Trastuzumab (anti-HER2 antibody) responsive and resistant breast cancer cells were injected into 48 hour-post-fertilization zebrafish embryos. Tumors were established for 24 hours and then fish were treated with a panel of breast cancer drugs and drug combinations. Autofluorescence lifetime images with single-cell resolution of the fish tumors were acquired at 24, 48, and 72 hours of drug treatment. Wide-field mCherry fluorescence and bright-field images with fish-level resolution were acquired prior to treatment (t=0 hour) and at 72 hours of drug treatment. Individual tumor responses were determined from the whole-fish bright-field and mCherry fluorescence images. Substantial differences in autofluorescence lifetime features, including optical redox ratio and mean NADH lifetime, were found between drug responsive and resistant tumors. These results suggest autofluorescence lifetime imaging is predictive of anti-cancer drug response in zebrafish xenografts
The heterogeneity and dynamic nature of cancerous tumors, such as those seen in breast cancer, pose a unique challenge in determining treatment regimens. The use of zebrafish as an in vivo model of breast cancer provides a high-throughput model with the potential to screen for efficacious drugs on a patient-by-patient basis. In this study, we use two-photon microscopy to measure metabolic changes in zebrafish with xenografted breast cancer tumors before, during, and after treatment with the anti-cancer drug paclitaxel. We use this metabolic imaging data to evaluate the zebrafish as a robust in vivo model of breast cancer. Preliminary results suggest the xenograft tumors respond to treatment with paclitaxel at 48 hours post treatment, as demonstrated by significant changes in NAD(P)H fluorescence lifetimes.
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