Varying label density allows artifact-free analysis of membrane-protein nanoclusters.

We present a method to robustly discriminate clustered from randomly distributed molecules detected with techniques based on single-molecule localization microscopy, such as PALM and STORM. The approach is based on deliberate variation of labeling density, such as titration of fluorescent antibody, combined with quantitative cluster analysis, and it thereby circumvents the problem of cluster artifacts generated by overcounting of blinking fluorophores. The method was used to analyze nanocluster formation in resting and activated immune cells.

Non-destructive characterization of adult zebrafish models using Jones matrix optical coherence tomography.

The zebrafish is a valuable vertebrate animal model in pre-clinical cancer research. A Jones matrix optical coherence tomography (JM-OCT) prototype operating at 1310 nm and an intensity-based spectral-domain OCT setup at 840 nm were utilized to investigate adult wildtype and a tumor-developing zebrafish model. Various anatomical features were characterized based on their inherent scattering and polarization signature. A motorized translation stage in combination with the JM-OCT prototype enabled large field-of-view imaging to investigate adult zebrafish in a non-destructive way. The diseased animals exhibited tumor-related abnormalities in the brain and near the eye region. The scatter intensity, the attenuation coefficients and local polarization parameters such as the birefringence and the degree of polarization uniformity were analyzed to quantify differences in tumor versus control regions. The proof-of-concept study in a limited number of animals revealed a significant decrease in birefringence in tumors found in the brain and near the eye compared to control regions. The presented work showed the potential of OCT and JM-OCT as non-destructive, high-resolution, and real-time imaging modalities for pre-clinical research based on zebrafish.

Multicontrast investigation of in vivo wildtype zebrafish in three development stages using polarization-sensitive optical coherence tomography.

SIGNIFICANCE: The scattering and polarization characteristics of various organs of in vivo wildtype zebrafish in three development stages were investigated using a non-destructive and label-free approach. The presented results showed a promising first step for the usability of Jones-matrix optical coherence tomography (JM-OCT) in zebrafish-based research. AIM: We aim to visualize and quantify the scatter and polarization signatures of various zebrafish organs for larvae, juvenile, and young adult animals in vivo in a non-invasive and label-free way. APPROACH: A custom-built polarization-sensitive JM-OCT setup in combination with a motorized translation stage was utilized to investigate live zebrafish. Depth-resolved scattering (intensity and attenuation coefficient) and polarization (birefringence and degree of polarization uniformity) properties were analyzed. OCT angiography (OCT-A) was utilized to investigate the vasculature label-free and non-destructively. RESULTS: The scatter and polarization signatures of the zebrafish organs such as the eye, gills, and muscles were investigated. The attenuation coefficient and birefringence changes between 1- and 2-month-old animals were evaluated in selected organs. OCT-A revealed the vasculature of in vivo larvae and juvenile zebrafish in a label-free manner. CONCLUSIONS: JM-OCT offers a rapid, label-free, non-invasive, tissue specific, and three-dimensional imaging tool to investigate in vivo processes in zebrafish in various development stages.

Evaluating cellularity and structural connectivity on whole brain slides using a custom-made digital pathology pipeline.

AIMS: In brain research, the histopathological examination of coronar whole-brain slides provides important insights into spatial disease characteristics. Regarding brain tumor research, this enables visualization of tumor heterogeneity, infiltration patterns and the relationship with the surrounding brain parenchyma. The precise correlation between radiological imaging and post-mortem brains is of special interest. NEW METHOD: We developed a wide-field slide scanner, comprising a microscope, a high-precision remotely controllable x-y-stage, a camera and a computer workstation, for automatically scanning uncommonly large formats. We analyzed whole brain slides of three patients and constructed cellularity heatmaps and fiber tract maps using a custom-made image processing pipeline. RESULTS: The obtained cellularity heatmaps allow for distinguishing compact tumor (5714 ± 1786 cells/mm², mean ± standard deviation) from white matter (3581 ± 828 cells/mm²) and grey matter (2473 ± 716 cells/mm²). Compared to magnetic resonance imaging, the proposed histopathological work-up (i) reveals a larger zone of tumor infiltration around the compact tumor areas and (ii) shows how pre-existing tracts are displaced by the tumor bulk. Moreover, we highlight differences in the histological tumor growth pattern of two different radiological progression subtypes. COMPARISON WITH EXISTING METHOD(S): Compared to existing (commercial) solutions, our slide scanning solution is adaptable and cost-efficient. Moreover, we showcase potential clinical applications by mapping whole brain histology to magnetic resonance imaging. CONCLUSIONS: We herein provide instructions on how to (i) construct a custom-built slide scanner capable of scanning arbitrary slide formats, (ii) automatically evaluate the cell density and (iii) perform fiber tracking on whole brain slides.

Assessment of pathological features in Alzheimer's disease brain tissue with a large field-of-view visible-light optical coherence microscope.

We implemented a wide field-of-view visible-light optical coherence microscope (OCM) for investigating ex-vivo brain tissue of patients diagnosed with Alzheimer's disease (AD) and of a mouse model of AD. A submicrometer axial resolution in tissue was achieved using a broad visible light spectrum. The use of various objective lenses enabled reaching micrometer transversal resolution and the acquisition of images of microscopic brain features, such as cell structures, vessels, and white matter tracts. Amyloid-beta plaques in the range of 10 to 70 µm were visualized. Large field-of-view images of young and old mouse brain sections were imaged using an automated x-y-z stage. The plaque load was characterized, revealing an age-related increase. Human brain tissue affected by cerebral amyloid angiopathy was investigated and hyperscattering structures resembling amyloid beta accumulations in the vessel walls were identified. All results were in good agreement with histology. A comparison of plaque features in both human and mouse brain tissue was performed, revealing an increase in plaque load and a decrease in reflectivity for mouse as compared with human brain tissue. Based on the promising outcome of our experiments, visible light OCM might be a powerful tool for investigating microscopic features in ex-vivo brain tissue.