Autofluorescence lifetime flow cytometry with time-correlated single photon counting.
Kayvan SamimiOjaswi PasachheEmmanuel Contreras GuzmanJeremiah M RiendeauAmani A GilletteDan L PhamKasia J WiechDarcie L MooreMelissa C SkalaPublished in: bioRxiv : the preprint server for biology (2024)
Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375nm picosecond-pulsed diode laser operating at 50MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification ( i.e ., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5ms integration time per cell, resulting in a light dose of 2.65 J/cm 2 that is well below damage thresholds (25 J/cm 2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent vs. activated (CD3/CD28/CD2) primary human T cells, and quiescent vs. activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.
Keyphrases
- induced apoptosis
- single cell
- label free
- cell cycle arrest
- high resolution
- deep learning
- single molecule
- signaling pathway
- high throughput
- machine learning
- endothelial cells
- high speed
- cell death
- mass spectrometry
- photodynamic therapy
- neural stem cells
- low cost
- bone marrow
- convolutional neural network
- small molecule
- mesenchymal stem cells
- optical coherence tomography
- cell proliferation
- young adults
- fluorescence imaging
- protein protein
- loop mediated isothermal amplification