## 4 Discussion  
In this study, a newly developed novel microchip was used to quickly examine nanogranules of human platelets in an aqueous solution. We could identify \(\alpha\)-granules of platelets by negative staining and immunoelectron microscopy using this microchip. Due to the required sample volume being very  
Figure 4: Immunoelectron micrographs of isolated platelet granules in the microchip. (**A**) This picture shows that the 6-nm gold particle-labeled granules are \(\alpha\)-granules, because P-selectin is located on the surface of these granules. The other vesicles not significantly labeled by gold particles might be lysosomes or exosomes. (**B**) The gold particles labeled granules were shown in higher magnification. There are abundant gold particles located on the surface of \(\alpha\)-granules (indicated by arrows). Scale bar, 0.2 \(\mu\)m.  
small and the steps being simple, this microchip and technology are believed to have more applications for observing nanogranules in the biomedical field in the future. In the past, to observe the ultrastructure of biological specimens, investigators usually used TEM, scanning electron microscopy (SEM), or cryo-electron microscopy (Cryo-EM). The specimen had to be fixed, dehydrated, infiltrated, and embedded in resin to obtain ultrathin sections, and then the ultrastructure of the sample was examined under an electron microscope [1]. Using SEM, specimens do not need to go through the steps of embedding and sectioning, but they must be dried and coated with metal particles, and only the surface structure of the sample can be observed [30]. Cryo-EM fixes specimens and obtains ultrathin sections by rapid freezing, without the need to administer many chemicals, which can maintain the native ultrastructure of a specimen. However, the operation process and experimental equipment of Cryo-EM have strict requirements to protect the specimen and prevent artifacts [31,32]. The specimen preparation for this novel microchip is relatively simple and rapid to observe the ultrastructure compared with that for TEM, SEM, or CryoEM. In addition, the specimen needs to be fixed with chemicals or liquid nitrogen in existing used technologies, but the specimen in this microchip can maintain its native state in the buffer or aqueous solution and can be directly observed under an electron microscope. Currently, flow cytometry is a powerful technique used to detect and isolate specific cells or particles according to their physical and chemical characteristics [33]. Functional analyses of platelets or granules often use flow cytometry [34]. Flow cytometry uses light properties scattered from cells or granules for identification or quantitative measurements of physical properties. More properties of a cell or granule can be explored, and more labels, dyes, and antibodies can be used for multi-parametric analyses. Therefore, the use of flow cytometry for particle identification or separation requires a lot of time, and samples should be labeled with antibodies to obtain sufficient results. The novel K-kit microchip requires only a very small amount of sample to quickly identify different granules, and can be used to perform morphological observations and comparative quantitative analyses. We could achieve qualitative analysis by comparing the shape of granules. In addition, the size of the nanoparticles can be directly measured and quantitatively analyzed. In addition, when using this novel K-kit microchip, the amount of biological samples required is less than 10 \(\upmu\)L. Therefore, this novel microchip is very suitable for use if biological samples are very precious or difficult to obtain. In addition, the protocols from whole-blood collection, platelet-rich preparation, isolation of platelet granules, and then examination by electron microscope just took less than 6 h using the microchip in this study. This allows investigators engaged in platelet research to quickly confirm and analyze the types of granules in a preparation and then proceed with advanced research.