Identification of Ca-rich dense granules in human platelets using scanning transmission X-ray microscopy

Whole-mount (WM) platelet preparation followed by transmission electron microscopy (TEM) observation is the standard method currently used to assess dense granule (DG) deficiency (DGD). However, due to the electron-density-based contrast mechanism in TEM, other granules such as α-granules might cause false DG detection. Here, scanning transmission X-ray microscopy (STXM) was used to identify DGs and minimize false DG detection of human platelets. STXM image stacks of human platelets were collected at the calcium (Ca) L_2,3 absorption edge and then converted to optical density maps. Ca distribution maps, obtained by subtracting the optical density maps at the pre-edge region from those at the post-edge region, were used to identify DGs based on the Ca richness. DGs were successfully detected using this STXM method without false detection, based on Ca maps for four human platelets. Spectral analysis of granules in human platelets confirmed that DGs contain a richer Ca content than other granules. The Ca distribution maps facilitated more effective DG identification than TEM which might falsely detect DGs. Correct identification of DGs would be important to assess the status of platelets and DG-related diseases. Therefore, this STXM method is proposed as a promising approach for better DG identification and diagnosis, as a complementary tool to the current WM TEM approach.