In vivo, two-color multiphoton microscopy using a femtosecond diamond Raman laser

Multiphoton microscopy is an essential tool for detailed study of neurovascular structure and function. Wavelength mixing of synchronized laser sources—two-color multiphoton microscopy—increases the spectral window of excitable fluorophores without the need for wavelength tuning. However, implementation of two-color microscopy requires a dual output laser source, which is typically costly and complicated. We have developed a relatively simple and low-cost diamond Raman laser pumped with a ytterbium fiber amplifier. The dual output system generates excitation light at both 1060 nm (pump wavelength) and 1250 nm (first Stokes emission of diamond laser) which, when temporally and spatially overlapped, yield an effective two-color excitation wavelength of 1160 nm. This source provides an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1060 nm excitation, twocolor excitation at 1160 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (e.g. tdKatushka2). We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color twophoton, and two-color three-photon microscopy in engineered 3-D multicellular spheroids. Additionally, we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation. This system can also be used to excite genetically encoded calcium indicators (e.g. RCaMP and GCaMP), which will be paramount in studying neuronal activity.