KEYWORDS: Autofluorescence, Diseases and disorders, Signal processing, Cancer, Two photon imaging, Receivers, Random forests, Optical imaging, Oncology, Modulation
T lymphocytes play a vital role in immune response, and as a result, their dysregulation is linked to numerous disease states. Imbalances of helper T cell subsets often precede symptomatic disease, indicating their potential as marker for disease detection and monitoring. Two-photon optical metabolic imaging (2P-OMI) offers advantages over conventional assays, as it can assess cellular metabolism on a single-cell level in a label-free manner. Preliminary data suggests 2P-OMI can identify helper T subtypes based solely on autofluorescence intensity and lifetime imaging of metabolic co-factors, potentially enabling rapid screening of patient immune cells to improve diagnoses and prognoses.
Autofluorescence lifetime imaging microscopy is sensitive to functional or treatment-induced metabolic changes in single cells. However, this label-free technique typically relies on laser-scanning microscopes that are not common for single-cell assessments in immunology. Here, we present a microfluidic lifetime-sensitive flow cytometer that measures time-domain decays of the metabolic co-enzyme NAD(P)H from single cells with picosecond temporal resolution using time-correlated single-photon counting (TCSPC) at a throughput of tens of cells per second. The system uses a picosecond-pulsed ultraviolet diode laser, alkali photomultiplier tubes, an FPGA-based time tagger, and provides real-time phasor-based classification (i.e., gating) of flowing cells for applications in immunology.
SignificanceAdvancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging).AimThis perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs.ApproachThree key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended.ConclusionImprovements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.
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