Fluorescence Nanoscopy of Platelets Resolves Platelet‐State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications

Abstract

Abstract:Dysregulation of how platelets store, sequester and release specific proteins seems to be implicated in many disease states, including cancer. Dual-color immunofluorescence stimulated emission depletion (STED) microscopy with 40 nm resolution was used to map pro-angiogenic VEGF, anti-angiogenic PF-4 and fibrinogen in more than 300 individual platelets. This imaging technique, offering distinct resolving advantages for protein localization studies in platelets, reveals that these proteins are stored in a segmented, zonal manner within regional clusters, significantly smaller than the size of an -granule. No co-localization between the different proteins could be observed. Moreover, upon platelet activation by thrombin or ADP, the proteins were found to undergo significant spatial rearrangements, including alterations in the size and number of the protein clusters, which were specific for a certain protein and the type of activation induced. Following these observations, we devised a simple assignment procedure, showing that the three distinct states of platelets (non-, ADP-and thrombin-activated) can be identified based on the average size, number and peripheral localization profiles of the regional protein clusters within the platelets. This suggests that high-resolution spatial mapping in platelets of specific proteins is a useful procedure to detect and characterize deviations in the selective storage, release and uptake of these proteins in the platelets. Since these deviations seem to be specific for, and may even underlie certain patophysiological states including cancer, these findings may have interesting diagnostic and even therapeutic implications.3

Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets

“…26 The number and size was measured computationally by locating the peak intensity of the individual emission profiles and then calculating their size as the full width at half maximum (FWHM) value. The peripheral distribution was measured using the actin image of the platelets to provide a mask of the total area spanned by the platelet.…”

“…The detailed fixation and immunofluorescence staining protocol can be found in reference. 26 STED-microscopy A custom built STED microscope was used for imaging, the principal design of which has been described in detail before. 35 The STED microscope can perform dual color imaging by having two separate excitation beams (570 ± 5 and 647 ± 5 nm), two STED beams (710 ± 10 and 750 ± 10 nm) and two emission pathways (615 ± 15 and 675 ± 15 nm).…”

Super-resolution microscopy in the diagnosis of platelet granule disorders

“…A recent paper used a different super-resolution technique, STED (see Box 2), to image the distribution of a number of proteins which are all believed to be located in the α-granules [5]. STED has a higher resolution than SIM (as fine as ~20 nm compared to ~120 nm) but is typically somewhat slower to acquire an image [15].…”

“…They are traditionally divided into dense (or δ-) granules and α-granules [2]. Recent work has suggested that this binary division is overly simplistic, identifying an additional granule type [4] and apparent subdivisions within α-granules [5]. Dense granules get their name for their appearance in whole mount transmission electron micrographs; that is to say that they are electron dense (due to the presence of a high concentration of calcium).…”

STED microscopy: increased resolution for medical research?

“…The profound reorganization of the cytoskeleton of cells, driven by a deregulation of signal transduction pathways as a response to changes in the extracellular environment, suggests the possibility of visualizing a correlation with tumourigenesis using STED . Additionally, the nanoscale topology of protein expression in blood platelets was imaged with STED microscopy, providing a means for clinical cancer diagnostics .…”