Expansion microscopy allows quantitative characterization of structural organization of platelet aggregates.

BACKGROUND

Current microscopy approaches applied to platelet aggregates from multiple settings indicate their structure has important implications for efficient hemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to detailed volumetric imaging of platelet aggregate structures. This is due to the small size of individual platelets and tight packing of platelets within aggregates, resulting in optical opacity.

OBJECTIVES

To address this, we investigated methods of performing superresolution microscopy and image analysis on aggregates of platelets to describe their morphology.

METHODS

We applied expansion microscopy and custom analysis workflows to extract quantitative information on the structure of platelet aggregates.

RESULTS

We demonstrated that expansion microscopy applied to platelet aggregates revealed multiscale information about their structure. We produced volumetric images at nanoscale resolution of >700 platelet aggregates stained for cytoskeletal and membrane components under normal conditions and following cytoskeletal disruption. We demonstrated that our custom analysis workflow provided quantitative description of platelet numbers, volumes, and morphology within entire platelet aggregates. Additionally, we quantitatively described subcellular organization of F-actin. By comparing these measurements following treatment with actin inhibitors, cytochalasin D, and latrunculin A, we could robustly detect structural disruptions in platelet aggregates.

CONCLUSION

Together, these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to subcellular features within individual platelets). This provides an important tool for analysis of platelet aggregates and thrombi in conditions such as hemostasis, immunothrombosis, and cardiovascular disease.