Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model

Abstract

We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The indepth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and-curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples. We demonstrated the biological importance of 2CALM, comparing the protein distributions of CD41 and CD62p on activated platelets in a 3D artificial clot. Additionally, using 2CALM, we quantified the impact of the inflammatory cytokine interleukin-1β on platelet activation in clots. The platform is applicable to any other cell type and biological system and can provide new insights into biological and medical applications.

Dual Channel Microfluidics for Mimicking the Blood–Brain Barrier

“…The astigmatism of the signal was used to determine the axial arrangement of the cytoskeleton. 43 , 44 The refractive index mismatch between the polymer structure and the cells was adjusted using a glucose buffer ( n ∼ 1.42). Figure 4 b shows a super-resolved (3D-dSTORM 45 ) image of the cytoskeleton.…”

“…To prove the microfluidic device’s feasibility for localization microscopy, we stained the cytoskeleton of these CD34 + -ECs with Alexa Fluor 647 conjugated phalloidin. The astigmatism of the signal was used to determine the axial arrangement of the cytoskeleton. , The refractive index mismatch between the polymer structure and the cells was adjusted using a glucose buffer ( n ∼ 1.42). Figure b shows a super-resolved (3D-dSTORM) image of the cytoskeleton.…”

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis

“…Afterwards, the cells were fixed and imaged at the single-molecule level. For the visualization of the EV distribution inside a cell, 3D reconstruction of astigmatism distorted single-molecule emitter point spread functions was used [ 54 , 56 , 57 , 59 , 60 ]. A high signal–noise ratio (on average >30) enabled the 3D localization of the subdiffractional EVs and aggregates inside the cells.…”

“…at room temperature and the unbound EVs were removed by four PBS washes. Imaging was performed on a modified Olympus IX81 inverted epifluorescence microscope with an oil-immersion objective lens (UApo N 100x/1.49 NA, Olympus, Vienna, Austria) [ 60 , 68 ]. A tube-lens with an additional magnification of 1.6 was used to achieve a final imaging magnification of 160 (corresponding to a pixel size of 100 nm).…”

Multi-level analysis of adipose tissue reveals the relevance of perivascular subpopulations and an increased endothelial permeability in early-stage lipedema

“…The following filter sets were used: dichroic filter (ZT405/488/561/640rpc, Chroma, Olching, Germany) and emission filters: ET 700/75 M (Chroma Technology GmbH) and ET 525/50 M (Chroma Technology GmbH, Olching, Germany). The laser illumination protocols, the camera and the stage are controlled by a custom-written software called QStdControl (C++, Qt framework) [29, 30]. The images were processed with Fiji (ImageJ 1.53c).…”