A single photon-excited confocal whole-cell autofluorescence lifetime system is described that uses fast field-programmable gate array (FPGA)-based time tagging electronics to implement time-correlated single photon counting (TCSPC) with simultaneous near-IR brightfield imaging. This system resolves variations in the fluorescence decay of the metabolic coenzyme NAD(P)H that provides high accuracy and precision for classifying activated and quiescent primary human T cells (ROC AUC = 0.98). This performance is similar to that obtained using commercial two-photon fluorescence lifetime imaging microscopy (FLIM). The small footprint, low cost, and high acquisition speed make this system attractive for monitoring cell biomanufacturing.
Current methods to assess T cell function use labels that prevent non-destructive quality control of T cell infusions. Here, we use autofluorescence imaging of NAD(P)H and FAD, co-enzymes of metabolism, in quiescent and activated T cells for label-free, non-destructive determination of T cell activation state and subtype. Logistic regression models achieved 97-99% accuracy for classification of T cell activation, and random forest models of achieved >97% accuracy for four-group classification of quiescent and activated CD3+CD8+ and CD3+CD4+ T cells. These results indicate that NAD(P)H and FAD imaging is a powerful method for label-free, non-destructive quality control of T cell infusions.
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.
Multiphoton laser scanning microscopy (MPLSM) utilizing techniques such as multiphoton excitation (MPE), second harmonic generation (SHG), and multiphoton fluorescence lifetime imaging and spectral lifetime imaging (FLIM and SLIM, respectively) are greatly expanding the degree of information obtainable with optical imaging in biomedical research. The application of these nonlinear optical approaches to the study of breast cancer holds particular promise. These noninvasive, multidimensional techniques are well suited to image exogenous fluorophores that allow relevant questions regarding protein localization and signaling to be addressed both in vivo and in vitro. Furthermore, MPLSM imaging of endogenous signals from collagen and fluorophores such as nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD), address important questions regarding the tumor-stromal interaction and the physiologic state of the cell. We demonstrate the utility of multimodal MPE/SHG/FLIM for imaging both exogenous and/or endogenous fluorophores in mammary tumors or relevant 3-D systems. Using SLIM, we present a method for imaging and differentiating signals from multiple fluorophores that can have overlapping spectra via SLIM Plotter—a computational tool for visualizing and analyzing large spectral-lifetime data sets.
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