Light attenuation in thick biological tissues, caused by a combination of absorption and scattering, limits the penetration depth in multiphoton microscopy (MPM). Both tissue scattering and absorption are dependent on wavelengths, which makes it essential to choose the excitation wavelength with minimum attenuation for deep imaging. Although theoretical models have been established to predict the wavelength dependence of light attenuation in brain tissues, the accuracy of these models in experimental settings needs to be verified. Furthermore, the water absorption contribution to the tissue attenuation, especially at 1450 nm where strong water absorption is predicted to be the dominant contributor in light attenuation, has not been confirmed. Here we performed a systematic study of in vivo three-photon imaging at different excitation wavelengths, 1300 nm, 1450 nm, 1500 nm, 1550 nm, and 1700 nm, and quantified the tissue attenuation by calculating the effective attenuation length at each wavelength. The experimental data show that the effective attenuation length at 1450 nm is significantly shorter than that at 1300 nm or 1700 nm. Our results provide unequivocal validation of the theoretical estimations based on water absorption and tissue scattering in predicting the effective attenuation lengths for long wavelength in vivo imaging.

Behaviors emerge from activity throughout the brain, but non-invasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become non-invasively accessible from embryo to sexually mature adult in a vertebrate model.

We demonstrate adaptive optics system based on nonlinear feedback from 3- and 4-photon fluorescence. The system is based on femtosecond pulses created by soliton self-frequency shift of a 1550-nm fiber-based femtosecond laser together with micro-electro-mechanical system (MEMS) phase spatial light modulator (SLM). We perturb the 1020-segment SLM using an orthogonal Walsh sequence basis set with a modified version of three-point phase shifting interferometry. We show the improvement after aberrations correction in 3-photon signal from fluorescent beads. In addition, we compare the improvement obtained in the same adaptive optical system for 2-, 3- and 4-photon fluorescence using dye pool. We show that signal improvement resulting from aberration correction grows exponentially as a function of the order of nonlinearity.

Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its non-invasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that 1300 nm and 1700 nm are the two best wavelength windows for deep brain imaging. Here, we combined long-wavelength illumination of ~1700 nm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with ~1micrometer spatial resolution in adult mouse brains, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added to any laser-scanning microscopy with simple and low-cost sources and detectors, such as continuous-wave diode lasers and InGaAs photodiodes. The long-wavelength, reflectance confocal imaging we demonstrated is label-free, and requires low illumination power. Furthermore, the imaging system is simple and low-cost, potentially creating new opportunities for biomedical research and clinical applications.

Spectral processor based on arrayed waveguide grating and free-space manipulation is capable of arbitrary filtering at record metrics of 0.8GHz resolution over 200GHz span. Narrowband coherent drop-demultiplexing and controlled optical shaping is demonstrated in unison with digital sub-banding. IntroductionLinear optical signal processing operations can assist optical communications in performing myriads of operations such as wavelength multiplexing, wavelength selective switching (WSS), and complex spectral filtering for channel selection, dispersion compensation, and signal shaping. Adaptive filtering operations are particularly of interest, enabling tuning of the center wavelength, bandwidth, dispersion compensation level and signal formats. Performing these operations with a spatial light modulator (SLM) operating on spatially dispersed light has been demonstrated [1-3]. These and similar experiments are based on a WSS platform, using a dispersive free-space optics arrangement with a bulk diffraction grating and lenses, and replacing a micro-mirror SLM [4] with a liquid-crystal on silicon (LCoS) SLM [5]. The performance of these instruments is set by the spectral resolution of the dispersing arrangement (typically 8-10GHz) and the pixel pitch determining spectral addressability (few GHz). Improved performance metrics can be obtained by replacing the bulk diffraction grating with an arrayed waveguide grating (AWG) [6,7], which is designed to provide higher dispersion values over a finite free spectral range (FSR). In this work we demonstrate ×10 improvement in the photonic spectral processor performance,