Since its first demonstrations, two-photon microscopy has enabled discoveries in many fields of biology since it is a uniquely suited imaging method for live / deep fluorescence imaging of biological tissues with sub-cellular resolution. However, imaging depth remains a crucial limitation for investigating scattering tissues such as the brain. To overcome this limitation and access deeper areas, one recent and very promising approach consists in using three-photon excitation (3PE) and shifting the excitation to the SWIR (Short-Wavelength InfraRed) range. Indeed, scattering decreases with increasing wavelength, following a typical 1/λ3 law. However water absorption should be avoided to prevent tissue from heating, so that it was realized recently that two optimal spectral excitation windows for deep multiphoton imaging lie in the 1300 and 1700 nm regions as shown in fig. 1 . We are presenting here the first dual-color SWIR source optimized for 3PE microscopy. The source is based on an OPCPA (optical parametric chirped pulse amplification) injected by a high-power Yb-fiber system. It operates at 1.25 MHz and emits concomitantly the two optimal 1.3 and 1.7 μm wavelengths with 69 fs, 65 fs pulse durations and with energies of 710 nJ and 3100 nJ respectively. These characteristics enabled us to record dual-color 3PE microscopy images in chick spinal cord, and in zebrafish and mouse brain.
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