Fluorescence laser-scanning microscopy (FLSM) is a widely utilized tool in life-science research. In recent years, this technique has undergone a profound transformation, thanks to the introduction of novel single-photon avalanche diode (SPAD) array detectors.
This study reveals the exciting possibilities of combining the SPAD array detector with single-molecule techniques.
We propose a real-time single-molecule tracking architecture, where the SPAD array effortlessly localizes the molecule of interest, and the beam scanning architecture effectively maintains the molecule at the center of the microscope's detection volume. This approach enables comprehensive three-dimensional tracking throughout the entire cell, offering valuable insights into molecular nano-environments, interactions, and structural changes through fluorescence lifetime information.
Furthermore, utilizing the same FLSM system, we present a novel sequential structure illumination single-molecule localization microscope (similar to MINFLUX). This advanced technique achieves localization precision in the few-nanometer range while simultaneously providing the molecule's fluorescence lifetime.
We present a high spectral resolution multiplexing acquisition mode for SRS microscopy based on a dual-beam femtosecond laser. A multi-channel Acousto Optical Tunable Filter (AOTF) generates spectral masks, given by the Hadamard matrix, by turning on and off different subsets of its 8 independent channels, corresponding to different wavelengths available within the broad bandwidth of the “pump” femtosecond laser. The SRS spectrum is retrieved by using the inverse Hadamard matrix. When additive noise is dominating, spectral measurements using the multiplexed method show the same signal to noise ratio of conventional single-wavenumber acquisitions performed with 4 times longer integration time.
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