Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy

Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy

Evaluation of the relationship between lacunar wall strain and osteocyte β-catenin signaling pathway activation using finite element methods and confocal fluorescence imaging.

Title from PDF of title page, viewed December 18, 2023Dissertation advisor: Thiagarajan GaneshVitaIncludes bibliographical references (pages 90-97)Dissertation (Ph.D)--Department of Civil and Mechanical Engineering, Department of Oral Biology and Craniofacial Sciences. University of Missouri--Kansas City, 2023Mechanical loading of bone has been shown to result in activation of Wnt/β-catenin signaling in osteocytes, leading to increased bone formation, but the mechanism of mechanotransduction is still unknown. It has been observed that following cyclic compression loading, osteocytes in murine cortical bone have heterogeneous levels of Wnt/β-catenin signaling. This led us to investigate whether the heterogeneity of bone strain at the lacunocanalicular walls might directly affect the level of Wnt/β-catenin signaling activity in an enclosed osteocyte. The right ulna of an anesthetized 5-mo male TOPGAL transgenic mouse was subjected to cyclic compression loading at 2.25 N at 2 Hz for 100 cycles and for control the left ulna was not loaded. After 1 hour both ulnae were harvested. Bone slices from the ulnae, were stained with the β-galactosidase (β-gal) substrate DDAOG to create a fluorescent stain that indicates the level of Wnt/β-catenin signaling activity in osteocytes. We labeled the lacunocanalicular network in the same samples with the fluorescent dye FITC. Multiplexed 3D confocal fluorescence images were collected, and the computational finite element modeling software, Materialise® Innovation Suites (MIMICs and 3-Matic modules), was used to assess the level of Wnt/β-catenin signaling in the osteocytes and generate finite element models of the bone with its lacunocanalicular network. The finite element analysis software FEBio was used to perform strain analysis on the models and predict bone strain in the bone matrix of the samples. The levels of Wnt/β-catenin signaling in the loaded bone were significantly higher than in the non-loaded bone. In the non-loaded bone, every osteocyte showed small amounts of activity, possibly a basal level of signaling or the result of prior cage activity. The levels of β-catenin signaling pathway activation were heterogeneous, and robust linear regression analysis reported correlation between activation and the average strain in regions representing bone surrounding the osteocytes. When only high strain elements were considered, however, the significance level of the correlation between osteocyte activation and strain was reduced. This suggests that in addition to bone strains, there may be other factors that are critical in determining osteocyte activation levels.Introduction -- Literature Review -- Methods -- Results -- Discussion -- Conclusio

A new filter for spot extraction in N-dimensional biological imaging

National audienc

Microglial involvement in experience-driven pruning of miswired retinal projections in Ten-m3 deficient mice

Precise neural connectivity is vital for brain function. The template for this is set-up during development through a spatiotemporal array of genetically determined cues. Neural activity, in part regulated by experience, also plays an important role in establishing neural connectivity though. Of note, environmental enrichment (EE) – a protocol that enhances social, physical, and cognitive experience - can drive partial repair of the miswired visual pathway of Ten-m3 knock-out (KO) mice. Remarkably, six weeks of EE from birth drove pruning of the most miswired retinogeniculate terminals. This was underway at postnatal day (P)26/27, but a mechanism was lacking. Microglia, the brain’s immune cells, have established roles in structural plasticity. The aim of this thesis was to determine whether microglia are involved in the EE-driven corrective pruning occurring in Ten-m3 KO mice. It was hypothesised EE might drive microglia to engulf the most miswired terminals in a defined postnatal window. Results supported this hypothesis. EE-driven, localised microglial-reactivity was reliably observed at the corrective pruning site of P25 Ten-m3 KO mice with no comparable effect in wildtypes. This followed a clearly defined time course - commencing between P18 and P21, peaking at P25 and ceased by P30 - and was accompanied by evidence of targeted microglial engulfment of the most miswired terminals. Both effects temporally aligned with the EE-driven corrective pruning. An attempt to confirm causal microglial involvement was made by administering daily minocycline injections from P18-24 with EE from birth. While the EE-driven corrective pruning, microglial reactivity and engulfment in Ten-m3 KO mice all appeared blocked with minocycline, the vehicle injections had the same effect, suggesting the injection protocol itself was countering EEs beneficial effect. Thus, while this thesis provides strong evidence for microglial involvement, the effect of minocycline remains inconclusive