The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter, up to six subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and Förster resonance energy transfer fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.

荧光显微镜的多路复用能力受到宽荧光光谱宽度的严重限制。光谱成像提供了潜在的解决方案，但分散局部发射光谱的典型方法显着阻碍了可达到的吞吐量。在这里，我们展示了使用单个固定荧光发射检测带，通过声光可调滤光器对来自白灯的激发波长进行帧同步快速扫描，最多六个亚细胞目标，由大量光谱重叠的常见荧光团标记, 可以在具有低 (~1%) 串扰和高时间分辨率 (低至~10 ms) 的活细胞中同时成像。通过分离激发光谱来量化同一样品中不同荧光团的丰度的能力接下来使我们能够设计出新颖的，活细胞中双态和 Förster 共振能量转移荧光生物传感器的定量成像方案。因此，我们在量化线粒体基质 pH 值和细胞内大分子拥挤方面实现了高灵敏度和时空分辨率，并首次进一步证明了绝对 pH 成像与三个额外的目标细胞器/蛋白质的多路复用，以阐明复杂的 Parkin 介导的线粒体自噬途径。总之，激发光谱显微镜为高度多路复用的荧光成像提供了绝佳的机会。无需荧光色散或关注检测器的光谱响应即可获取快速光谱图像的前景提供了巨大的潜力。