Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser

Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning. However, most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach. In this report, we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm, which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs. The two excitation wavelengths of 1055 and 1240 nm, along with the effective two-color excitation wavelength of 1140 nm, provide an almost complete coverage of fluorophores excitable within the range of 1000–1300 nm. When compared with 1055 nm excitation, two-color excitation at 1140 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (for example, tdKatushka2). We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color two-photon, and two-color three-photon microscopy in engineered 3D multicellular spheroids. We further discuss potential benefits and applications for two-color three-photon excitation. In addition, 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. A diamond Raman laser pumped by a ytterbium-fiber amplifier is a convenient and stable optical source for multiphoton imaging. Andrew Dunn and his co-workers from the University of Texas at Austin in the USA and Macquarie University in Australia used the combined outputs from a diamond Raman laser at 1240 nanometers and a ytterbium-fiber amplifier at 1055 nanometers to perform both two-color, two-photon and two-color, three-photon imaging of biological samples. In particular, they demonstrated multicolor microscopy of cells labeled with a far-red fluorescent protein and a blue-emitting stain. In addition, deep in vivo microscopy of the vasculature in the brain of an adult mouse perfused with a red dye was performed at cortex depths of up to 1 millimeter using the system for two-color, two-photon excitation.