Thrombosis remains a global health concern, necessitating research into its underlying mechanisms. Utilizing a high-speed bright-field microscope based on optical frequency-division multiplexing and microfluidics, we performed image-based single-cell profiling and temporal monitoring of circulating platelet aggregates that are the precursors to thrombosis. Our analysis encompassed 41 thrombosis patients, 110 COVID-19 patients, and 11 healthy individuals. By investigating the morphological changes of platelet aggregates under the influence of thrombosis, COVID-19, and COVID-19 vaccination, we observed distinct morphological alterations in platelet aggregates across different conditions, which shed light on the interplay between platelet aggregation and thrombotic events.
There is widespread concern about the safety of COVID-19 vaccinations related to platelet hyperactivity. However, their long-term influence on platelet activity remains unknown. We address this issue by applying a high-speed bright-field microscope based on optical frequency-division multiplexing and microfluidics for massive image-based analysis. We performed image-based single-cell profiling and temporal monitoring of circulating platelet aggregates in the blood samples of healthy human participants before and after they received three vaccination doses over a nearly one-year period. The results demonstrate no significant or persistent change in platelet activity after vaccine doses.
We demonstrate ultrafast fluorescence lifetime imaging microscopy (FLIM) based on frequency-division multiplexing. As a proof-of-concept demonstration, we obtained images with fluorescence intensity and lifetime contrasts of MCF-7 breast cancer cells stained by SYTO16 at a record high frame rate of 16,000 fps, which is 100 times higher than that of previously reported FLIM techniques. Our method is expected to expand the utility of FLIM to quantitative analysis of rapid intracellular dynamics and high-throughput cell screening based on fluorescence lifetime images.
Frequency-division-multiplexed laser-scanning fluorescence microscopy is a powerful imaging method for biological tissues that enables an imaging speed of >10,000 frames/s. Despite its unprecedented high speed, its large-scale implementation that includes a bulky and unstable Mach-Zehnder interferometer has hampered its practical applications, especially in biomedical studies. Here we present a compact implementation of frequency-division-multiplexed microscopy to overcome this issue. The compactness is enabled by introducing an inline interferometer for generating an excitation beam array. In this setup, the laser beam is separated and recombined with small beam separation angles (<2°) by optical components such as acousto-optic deflectors or Wollaston prisms, thus implementing an interferometer with a relay lens system and drastically downsizing the setup. Compared with our previous setup with a Mach-Zehnder interferometer, the footprint of the optical setup for the excitation beam generation was downsized from ~20 cm x 70 cm to ~130 cm x 2.54 cm (defined by one-inch optical components used in the setup). Furthermore, our design concept allows for an ultra-compact implementation (~10 cm x 1 cm) by using custom optical components and omitting the relay lens systems. As a proof-of-concept demonstration, we obtained two-color (fluorescence and brightfield) images of Euglena gracilis cells (autofluorescent) and MCF-7 cells (fluorescence from nuclei stained by SYTO16) at a scanning speed of 0.84 m/s, which corresponds to a frame rate of 15,300 frames/s at a 55-μm field of view in the scanning direction. By virtue of the wide modulation bandwidth of the excitation beam (200 MHz), it is also possible to measure fluorescence lifetimes of target fluorophores, leading to potential applications for fluorescence lifetime imaging (FLIM).
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