Lightweight and head-mountable scanning nonlinear fiberscope technologies offer an exciting opportunity for enabling mechanistic exploration of ensemble neural activities with subcellular resolution on freely behaving rodents. The tether of the fiberscope, consisting of an optical fiber and scanner drive wires, however, restricts the mouse’s movement and consequently precludes free rotation and limits the freedom of walking. Here we present the first twist-free two-photon fiberscope technology for enabling neuroimaging on freely rotating/walking mice. The technology equips a scanning fiberscope with active rotational tracking and compensation capabilities through an optoelectrical commutator (OEC) to allow the animal to rotate and walk in arbitrary patterns during two-photon fluorescence (TPF) imaging of neural activities. The OEC provides excellent optical coupling stability ( < ± 1 % fluctuation during rotation) and an extremely high torque sensitivity ( < 8 m N ⋅ m ). In addition, the new technology is equipped with a custom grating and prism to effectively manage the temporal properties of the femtosecond excitation pulses through the fiber-optic system, which improved neuroimaging signal by more than 2 X . This TPF fiberscope imaging platform has been tested for in vivo imaging, and the results demonstrate that it enables reliable recording of calcium dynamics of more than 50 neurons simultaneously in the motor cortices of freely behaving mice in a twist-free fashion. With active tracking function of the OEC enabled, we observed considerable increase in both behavior and neural activities in the motor cortices of the mice during freely behaving neuroimaging experiments.

Fiber-optic-based two-photon fluorescence endomicroscopy is emerging as an enabling technology for in vivo histological imaging of internal organs and functional neuronal imaging on freely-behaving animals. However, high-speed imaging remains challenging due to the expense of miniaturization and lack of suited fast beam scanners. For many applications, a higher imaging speed is highly desired, especially for monitoring functional dynamics such as transient dendritic responses in neuroscience. This Letter reports the development of a fast fiber-optic scanning endo-microscope with an imaging speed higher than 26 frames/s. In vivo neural dynamics imaging with the high-speed endomicroscope was performed on a freely-behaving mouse over the primary motor cortex that expressed GCaMP6m. The results demonstrate its capability of real-time monitoring of transient neuronal dynamics with very fine temporal resolution.

Compactness, among several others, is one unique and very attractive feature of a scanning fiber-optic two-photon endomicroscope. To increase the scanning area and the total number of resolvable pixels (i.e., the imaging throughput), it typically requires a longer cantilever which, however, leads to a much undesired, reduced scanning speed (and thus imaging frame rate). Herein we introduce a new design strategy for a fiber-optic scanning endomicroscope, where the overall numerical aperture (NA)or beam focusing power is distributed over two stages: 1) a mode-field focuser engineered at the tip of a double-clad fiber (DCF) cantilever to pre-amplify the single-mode core NA, and 2) a micro objective of a lower magnification (i.e., ~2× in this design) to achieve final tight beam focusing. This new design enables either an ~9-fold increase in imaging area (throughput) or an ~3-fold improvement in imaging frame rate when compared to traditional fiber-optic endomicroscope designs. The performance of an as-designed endomicroscope of an enhanced throughput-speed product was demonstrated by two representative applications: (1) high-resolution imaging of an internal organ (i.e., mouse kidney) in vivo over a large field of view without using any fluorescent contrast agents, and (2) real-time neural imaging by visualizing dendritic calcium dynamics in vivo with sub-second temporal resolution in GCaMP6m-expressing mouse brain. This cascaded NA amplification strategy is universal and can be readily adapted to other types of fiber-optic scanners in compact linear or nonlinear endomicroscopes.

Objective/background: In vivo imaging and quantification of the microstructures of small airways in three dimensions (3D) allows a better understanding and management of airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). At present, the resolution and contrast of the currently available conventional optical coherence tomography (OCT) imaging technologies operating at 1300 nm remain challenging to directly visualize the fine microstructures of small airways in vivo . Methods: We developed an ultrahigh-resolution diffractive endoscopic OCT at 800 nm to afford a resolving power of 1.7 μm (in tissue) with an improved contrast and a custom deep residual learning based image segmentation framework to perform accurate and automated 3D quantification of airway anatomy. Results: The 800-nm diffractive OCT enabled the direct delineation of the structural components in the small airway wall in vivo . We further first demonstrated the 3D anatomic quantification of critical tissue compartments of small airways in sheep using the automated segmentation method. Conclusion: The deep learning assisted diffractive OCT provides a unique ability to access the small airways, directly visualize and quantify the important tissue compartments, such as airway smooth muscle, in the airway wall in vivo in 3D. Significance: These pilot results suggest a potential technology for calculating volumetric measurements of small airways in patients in vivo .

Real-time intraoperative delineation of cancer and non-cancer brain tissues, especially in the eloquent cortex, is critical for thorough cancer resection, lengthening survival, and improving quality of life. Prior studies have established that thresholding optical attenuation values reveals cancer regions with high sensitivity and specificity. However, threshold of a single value disregards local information important to making more robust predictions. Hence, we propose deep convolutional neural networks (CNNs) trained on labeled OCT images and co-occurrence matrix features extracted from these images to synergize attenuation characteristics and texture features. Specifically, we adapt a deep ensemble model trained on 5,831 examples in a training dataset of 7 patients. We obtain 93.31% sensitivity and 97.04% specificity on a holdout set of 4 patients without the need for beam profile normalization using a reference phantom. The segmentation maps produced by parsing the OCT volume and tiling the outputs of our model are in excellent agreement with attenuation mapping-based methods. Our new approach for this important application has considerable implications for clinical translation.

Vascular-targeted photodynamic therapy (VTP) is an emerging treatment for tumors. The change of tumor vasculatures, including a newly-formed microvascular, in response to VTP, is a key assessment parameter for optimizing the treatment effect. However, an accurate assessment of vasculature, particularly the microvasculature’s changes in vivo, remains challenging due to the limited resolution afforded by existing imaging modalities. In this study, we demonstrated the in vivo imaging of VTP effects on an A431 tumor-bearing window chamber model of a mouse with an 800-nm ultrahigh-resolution functional optical coherence tomography (UHR-FOCT). We further quantitatively demonstrated the effects of VTP on the size and density of tumor microvasculature before, during, and after the treatment. Our results suggest the promising potential of UHR-FOCT for assessing the tumor treatment with VTP in vivo and in real time to achieve an optimal outcome.