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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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