The broad use of two-photon microscopy has been enabled in part by Ti:Sapphire femtosecond lasers, which offer a wavelength-tunable source of pulsed excitation. Action spectra have thus been primarily reported for the tunable range of Ti:Sapphire lasers (∼700–1000 nm). However, longer wavelengths offer deeper imaging in tissue via reduced scattering and spectral dips in water absorption, and new generations of pulsed lasers offer wider tunable ranges. We present the peak molecular brightness spectra for eight Alexa Fluor dyes between 700–1300 nm as a first-order surrogate for action spectra measured with an unmodified commercial microscope, which reveal overlapping long-wavelength excitation peaks with potential for multiplexed excitation. We demonstrate simultaneous single-wavelength excitation of six spectrally overlapping fluorophores using either short (∼790 nm) or long (∼1090 nm) wavelengths, and that the newly characterized excitation peaks measured past 1000 nm offer improved photostability and enhanced fidelity of linear spectral unmixing at depth compared to shorter wavelengths.

中文翻译：

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双光子峰分子亮度光谱揭示了多路复用成像深度和光稳定性的长波长增强

Ti:Sapphire 飞秒激光器部分实现了双光子显微镜的广泛使用，该激光器提供了波长可调的脉冲激发源。因此，主要报道了 Ti:Sapphire 激光器的可调谐范围（~700-1000 nm）的作用光谱。然而，更长的波长通过减少水吸收的散射和光谱下降在组织中提供更深的成像，并且新一代脉冲激光器提供更宽的可调范围。我们提供了 700-1300 nm 之间的八种 Alexa Fluor 染料的峰值分子亮度光谱，作为用未经修改的商业显微镜测量的作用光谱的一级替代物，其揭示了具有多重激发潜力的重叠长波长激发峰。