Anatomical Modeling of Brain Vasculature in Two-Photon Microscopy by Generalizable Deep Learning

Objective and Impact Statement.Segmentation of blood vessels from two-photon microscopy(2PM)angiograms of brains has important applications in hemodynamic analysis and disease diagnosis.Here,we develop a generalizable deep learning technique for accurate 2PM vascular segmentation of sizable regions in mouse brains acquired from multiple 2PM setups.The technique is computationally efficient,thus ideal for large-scale neurovascular analysis.Introduction.Vascular segmentation from 2PM angiograms is an important first step in hemodynamic modeling of brain vasculature.Existing segmentation methods based on deep learning either lack the ability to generalize to data from different imaging systems or are computationally infeasible for large-scale angiograms.In this work,we overcome both these limitations by a method that is generalizable to various imaging systems and is able to segment large-scale angiograms.Methods.We employ a computationally efficient deep learning framework with a loss function that incorporates a balanced binary-cross-entropy loss and total variation regularization on the network’s output.Its effectiveness is demonstrated on experimentally acquired in vivo angiograms from mouse brains of dimensions up to 808×808×702μm.Results.To demonstrate the superior generalizability of our framework,we train on data from only one 2PM microscope and demonstrate high-quality segmentation on data from a different microscope without any network tuning.Overall,our method demonstrates 10×faster computation in terms of voxels-segmented-per-second and 3×larger depth compared to the state-of-the-art.Conclusion.Our work provides a generalizable and computationally efficient anatomical modeling framework for brain vasculature,which consists of deep learning-based vascular segmentation followed by graphing.It paves the way for future modeling and analysis of hemodynamic response at much greater scales that were inaccessible before.

Mid-infrared chemical imaging of intracellular tau fibrils using fluorescence-guided computational photothermal microscopy

Amyloid proteins are associated with a broad spectrum of neurodegenerative diseases.However,it remains a grand challenge to extract molecular structure information from intracellular amyloid proteins in their native cellular environment.To address this challenge,we developed a computational chemical microscope integrating 3D midinfrared photothermal imaging with fluorescence imaging,termed Fluorescence-guided Bond-Selective Intensity Diffraction Tomography(FBS-IDT).Based on a low-cost and simple optical design,FBS-IDT enables chemical-specific volumetric imaging and 3D site-specific mid-IR fingerprint spectroscopic analysis of tau fbrils,an important type of amyloid protein aggregates,in their intracellular environment.Label-free volumetric chemical imaging of human cells with/without seeded tau fibrils is demonstrated to show the potential correlation between lipid accumulation and tau aggregate formation.Depth-resolved mid-infrared fingerprint spectroscopy is performed to reveal the protein secondary structure of the intracellular tau fibrils.3D visualization of theβ-sheet for tau fibril structure is achieved.

Global profiling of O-GlcNAcylated and/or phosphorylated proteins in hepatoblastoma

O-linked-β-N-acetylglucosamine(O-GlcNAc)glycosylation(O-GlcNAcylation)and phosphorylation are critical posttranslational modifications that are involved in regulating the functions of proteins involved in tumorigenesis and the development of various solid tumors.However,a detailed characterization of the patterns of these modifications at the peptide or protein level in hepatoblastoma(HB),a highly malignant primary hepatic tumor with an extremely low incidence in children,has not been performed.Here,we examined O-GlcNAc-modified or phospho-modified peptides and proteins in HB through quantitative proteomic analysis of HB tissues and paired normal liver tissues.Our results identified 114 O-GlcNAcylated peptides belonging to 78 proteins and 3494 phosphorylated peptides in 2088 proteins.Interestingly,41 proteins were modified by both O-GlcNAcylation and phosphorylation.These proteins are involved in multiple molecular and cellular processes,including chromatin remodeling,transcription,translation,transportation,and organelle organization.In addition,we verified the accuracy of the proteomics results and found a competitive inhibitory effect between O-GlcNAcylation and phosphorylation of HSPB1.Further,O-GlcNAcylation modification of HSPB1 promoted proliferation and enhanced the chemotherapeutic resistance of HB cell lines in vitro.Collectively,our research suggests that O-GlcNAc-modified and/or phospho-modified proteins may play a crucial role in the pathogenesis of HB.