High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 50 neurons in the hippocampal stratum pyramidale at ~1-mm depth within an intact mouse brain. our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-μm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

The measured dispersion of a low-loss, hollow-core photonic band-gap fiber is anomalous throughout most of the transmission band, and its variation with wavelength is large compared with that of a conventional step-index fiber. For an air-filled fiber, femtosecond self-frequency--shifted fundamental solitons with peak powers greater than 2megawatts can be supported. For Xe-filled fibers, nonfrequency-shifted temporal solitons with peak powers greater than 5.5 megawatts can be generated, representing an increase in the power that can be propagated in an optical fiber of two orders of magnitude. The results demonstrate a unique capability to deliver high-power pulses in a single spatial mode over distances exceeding 200 meters.

We present a compact and flexible endoscope (3-mm outer diameter, 4-cm rigid length) that utilizes a miniaturized resonant/nonresonant fiber raster scanner and a multielement gradient-index lens assembly for two-photon excited intrinsic fluorescence and second-harmonic generation imaging of biological tissues. The miniaturized raster scanner is fabricated by mounting a commercial double-clad optical fiber (DCF) onto two piezo bimorphs that are aligned such that their bending axes are perpendicular to each other. Fast lateral scanning of the laser illumination at 4.1 frames∕s (512 lines per frame) is achieved by simultaneously driving the DCF cantilever at its resonant frequency in one dimension and nonresonantly in the orthogonal axis. The implementation of a DCF into the scanner enables simultaneous delivery of the femtosecond pulsed 800-nm excitation source and epi-collection of the signal. Our device is able to achieve a field-of-view (FOV xy ) of 110 μm by 110 μm with a highly uniform pixel dwell time. The lateral and axial resolutions for two-photon imaging are 0.8 and 10 μm, respectively. The endoscope's imaging capabilities were demonstrated by imaging ex vivo mouse tissue through the collection of intrinsic fluorescence and second-harmonic signal without the need for staining. The results presented here indicate that our device can be applied in the future to perform minimally invasive in vivo optical biopsies for medical diagnostics.nonlinear optical endoscopy | real-time optical diagnosis | scanning fiber endoscopy | microendoscopy | endogenous fluorescence M ultiphoton imaging techniques such as two-photon fluorescence (TPF) and second-harmonic generation (SHG) microscopy hold great promise for the future of medical diagnosis because of their potential to replace surgical biopsies with minimally invasive optical diagnosis of tissue health (1-7). In a clinical setting, these diagnostic techniques will be capable of acquiring real-time, high-resolution, in vivo images without the need for contrast agents. However, a challenge in translating these beneficial imaging technologies into the clinic lies in successfully miniaturizing bulky tabletop microscope components into a compact probe without degrading the overall imaging performance of the system. These microendoscopes would not only have the potential to be used as diagnostic tools capable of early cancer detection, but could also be used for such applications as photodynamic therapy and microsurgery (8,9).A number of groups have demonstrated miniaturized instruments capable of confocal, optical coherence tomography (OCT), TPF, and SHG imaging (10-25). The primary constituents of these devices are typically a miniaturized scanning mechanism and lens assembly that is encapsulated in a protective housing with dimensions suitable for minimally invasive procedures (i.e., a probe outer diameter on the order of a few millimeters with a rigid length of several centimeters). Within these microendoscopes, various distal miniaturized scanners have been demonstr...