Our study introduces a label-free imaging and quantitative analysis approach for investigating lipofuscin aggregates in human brain tissue. Leveraging the colocalization of lipofuscin with cell soma, our novel method accurately identifies and counts cells, especially large neurons. Achieving an impressive 92% accuracy at submicron resolution, our label-free approach outperforms the commonly used Nissl stain. We develop a robust segmentation technique for lipofuscin aggregates, revealing layered structures in the cortical gray matter, potentially associated with cell distribution. Furthermore, we validate our results using state-of-the-art techniques, including fluorescence lifetime imaging microscope and sub-micron resolution two photon imaging. Our findings contribute valuable insights into neurodegenerative diseases and hold promise for future diagnostic advancements.
Cerebral microvascular alterations are increasingly recognized as important contributors to neurodegenerative diseases, such as Alzheimer's Disease (AD) and chronic traumatic encephalopathy (CTE). To characterize these changes, we used a custom serial-sectioning, polarization-sensitive optical coherence tomography system to collect volumetric images of brain tissue volumes from the dorsolateral frontal lobe of normal controls and subjects with pathologically confirmed AD and CTE. We performed vasculature segmentation on each tissue volume and then analyzed the geometric properties of the vascular networks. The preliminary results suggest reduced microvascular density in AD compared to both CTE and NC.
SignificanceBrief disruptions in capillary flow, commonly referred to as capillary “stalling,” have gained interest recently for their potential role in disrupting cerebral blood flow and oxygen delivery. Approaches to studying this phenomenon have been hindered by limited volumetric imaging rates and cumbersome manual analysis. The ability to precisely and efficiently quantify the dynamics of these events will be key in understanding their potential role in stroke and neurodegenerative diseases, such as Alzheimer’s disease.AimOur study aimed to demonstrate that the fast volumetric imaging rates offered by Bessel beam two-photon microscopy combined with improved data analysis throughput allows for faster and more precise measurement of capillary stall dynamics.ResultsWe found that while our analysis approach was unable to achieve full automation, we were able to cut analysis time in half while also finding stalling events that were missed in traditional blind manual analysis. The resulting data showed that our Bessel beam system was captured more stalling events compared to optical coherence tomography, particularly shorter stalling events. We then compare differences in stall dynamics between a young and old group of mice as well as a demonstrate changes in stalling before and after photothrombotic model of stroke. Finally, we also demonstrate the ability to monitor arteriole dynamics alongside stall dynamics.ConclusionsBessel beam two-photon microscopy combined with high throughput analysis is a powerful tool for studying capillary stalling due to its ability to monitor hundreds of capillaries simultaneously at high frame rates.
Significance: Whether or not capillary pericytes contribute to blood flow regulation in the brain and retina has long been debated. This was partly caused by failure of detecting the contractile protein α-smooth muscle actin (α-SMA) in capillary pericytes.
Aim: The aim of this review is to summarize recent developments in detecting α-SMA and contractility in capillary pericytes and the relevant literature on the biology of actin filaments.
Results: Evidence suggests that for visualization of the small amounts of α-SMA in downstream mid-capillary pericytes, actin depolymerization must be prevented during tissue processing. Actin filaments turnover is mainly based on de/re-polymerization rather than transcription of the monomeric form, hence, small amounts of α-SMA mRNA may evade detection by transcriptomic studies. Similarly, transgenic mice expressing fluorescent reporters under the α-SMA promoter may yield low fluorescence due to limited transcriptional activity in mid-capillary pericytes. Recent studies show that pericytes including mid-capillary ones express several actin isoforms and myosin heavy chain type 11, the partner of α-SMA in mediating contraction. Emerging evidence also suggests that actin polymerization in pericytes may have a role in regulating the tone of downstream capillaries.
Conclusions: With guidance of actin biology, innovative labeling and imaging techniques can reveal the molecular machinery of contraction in pericytes.
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