Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0.

The limited per-pixel bandwidth of most microscopy methods requires compromises between field of view, sampling density and imaging speed. This limitation constrains studies involving complex motion or fast cellular signaling, and presents a major bottleneck for high-throughput structural imaging. Here, we combine high-speed intensified camera technology with a versatile, reconfigurable and dramatically improved Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design that can achieve high-resolution volumetric imaging at over 300 volumes per second and over 1.2 GHz pixel rates. We demonstrate near-isotropic sampling in freely moving Caenorhabditis elegans, and analyze real-time blood flow and calcium dynamics in the beating zebrafish heart. The same system also permits high-throughput structural imaging of mounted, intact, cleared and expanded samples. SCAPE 2.0's significantly lower photodamage compared to point-scanning techniques is also confirmed. Our results demonstrate that SCAPE 2.0 is a powerful, yet accessible imaging platform for myriad emerging high-speed dynamic and high-throughput volumetric microscopy applications.

Classifying multiple sclerosis patients on the basis of SDMT performance using machine learning.

OBJECTIVE: To build a model to predict cognitive status reflecting structural, functional, and white matter integrity changes in early multiple sclerosis (MS). METHODS: Based on Symbol Digit Modalities Test (SDMT) performance, 183 early MS patients were assigned "lower" or "higher" performance groups. Three-dimensional (3D)-T2, T1, diffusion weighted, and resting-state magnetic resonance imaging (MRI) data were acquired in 3T. Using Random Forest, five models were trained to classify patients into two groups based on 1-demographic/clinical, 2-lesion volume/location, 3-local/global tissue volume, 4-local/global diffusion tensor imaging, and 5-whole-brain resting-state-functional-connectivity measures. In a final model, all important features from previous models were concatenated. Area under the receiver operating characteristic curve (AUC) values were calculated to evaluate classifier performance. RESULTS: The highest AUC value (0.90) was achieved by concatenating all important features from neuroimaging models. The top 10 contributing variables included volumes of bilateral nucleus accumbens and right thalamus, mean diffusivity of left cingulum-angular bundle, and functional connectivity among hubs of seven large-scale networks. CONCLUSION: These results provide an indication of a non-random brain pattern mostly compromising areas involved in attentional processes specific to patients who perform worse in SDMT. High accuracy of the final model supports this pattern as a potential neuroimaging biomarker of subtle cognitive changes in early MS.

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.