Opt Lett. 2021 Jan 1;46(1):37-40. doi: 10.1364/OL.413526.
We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.
我们通过使用线照明和狭缝共焦检测的可见波长双光子激发显微镜展示了高光谱成像。使用 530nm 的飞秒脉冲激光同时激发具有不同发射波长的荧光蛋白。线照明的使用使得高效地检测高光谱图像成为可能，并在对活 HeLa 细胞的观察中实现了同时检测三个荧光光谱，每条线的曝光时间为 1ms，相当于点扫描的每个像素约 2µs，每个光谱有 160 个数据点。通过组合线性光谱解混技术，实现了细胞中荧光探针的定位。对成像特性的理论研究表明，通过非线性激发和狭缝检测的组合可以获得高深度分辨特性。
