Super-resolution imaging of platelet-activation process and its quantitative analysis

Abstract

Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.

Development of a New Approach for Low-Laser-Power Super-Resolution Fluorescence Imaging

“…For STORM imaging, the samples were incubated in an imaging buffer containing a thiol reagent [mercaptoethylamine (MEA) (30070; Sigma-Aldrich), 2mercaptoethanol (βME) (M6250; Sigma-Aldrich), or dithiothreitol (DTT) (R0861; Thermo Scientific)], an oxygen scavenger system [5% glucose (w/v), 0.5 mg/mL glucose oxidase (G2133; Sigma-Aldrich)], and 38 μg/mL catalase (C3515; Sigma-Aldrich) in D-PBS, which has a refractive index of 1.34 and pH 8.0. 19 To change the pH, sodium hydroxide or hydrochloric acid was added by measuring the pH using a pH meter (Starter 3100; OHAUS). To adjust the refractive index of the imaging buffer, we added up to 60% (w/v) sucrose in the imaging buffer as described, finally obtaining a refractive index of up to 1.45.…”

Visualizing extracellular vesicle biogenesis in gram-positive bacteria using super-resolution microscopy

“…These collected centroids were drift-corrected and rendered using several parameters for the final STORM image. For multi-color images, the images from two color channels of the same sample area were correlated using custom-written MATLAB code [ 13 ].…”

“…Currently, the observation and quantification of EV formation in gram-positive bacteria remains challenging due to the technical difficulties associated with imaging the ultrastructure of small-sized EVs (20-400 nm in diameter) within the diffraction-limited area using conventional light microscopy (LM) techniques [5]. Although electron microscopy (EM) can provide higher resolution than LM, the molecular-specific labeling efficiency is limited in EM images [13][14][15]. These difficulties can be overcome using recently developed super-resolution fluorescence microscopy, which enables nanoscale imaging with high molecular specificity [16][17][18][19].…”

Development of Deep-Learning-Based Single-Molecule Localization Image Analysis

“…For example, Chan et al reported purinosome transit in the context of subcellular localization using STORM imaging, which has not been resolved using conventional microscopy [6]. The mechanisms of organelle redistribution and sorting in platelet intermediate maturation and activation processes have also been investigated with the help of nanoscale imaging by STORM [20,21].…”