A Novel Microchip Technique for Quickly Identifying Nanogranules in an Aqueous Solution by Transmission Electron Microscopy: Imaging of Platelet Granules

Abstract

Ultrastructural observation of biological specimens or nanogranules usually requires the use of electron microscopy. Electron microscopy takes a lot of time, requires many steps, and uses many chemicals, which may affect the native state of biological specimens. A novel microchip (K-kit) was used as a specimen kit for in situ imaging of human platelet granules in an aqueous solution using a transmission electron microscope. This microchip enabled us to observe the native human platelet granules very quickly and easily. The protocols included blood collection, platelet purification, platelet granule isolation, sample loading into this microchip, and then observation by a transmission electron microscope. In addition, these granules could still remain in aqueous solution, and only a very small amount of the sample was required for observation and analysis. We used this microchip to identify the native platelet granules by negative staining. Furthermore, we used this microchip to perform immunoelectron microscopy and successfully label α-granules of platelets with the anti-P-selectin antibody. These results demonstrate that the novel microchip can provide researchers with faster and better choices when using a transmission electron microscope to examine nanogranules of biological specimens in aqueous conditions.

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“…Although SEM does not require embedding and sectioning, the sample must be dried and coated with metal particles for visualization of the surface structure. In this study, a newly developed novel microchip was used for quick examination of the changes of the P-selectin content in α-granules in human platelets, in which the microchip maintains its native state in the aqueous solution and can be observed directly under an electron microscope …”

“…In this study, a newly developed novel microchip was used for quick examination of the changes of the P-selectin content in α-granules in human platelets, in which the microchip maintains its native state in the aqueous solution and can be observed directly under an electron microscope. 16 In the current study, PTE (IC 50 : 2.5 μmol/L) strongly inhibited collagen-induced platelet aggregation, which suggests that PTE mediated a distinct PLC-dependent mechanism. The PLCγ family consists of isozymes 1 and 2, and PLCγ2 participates in collagen-dependent signaling in platelets.…”

“…Immunoelectron microscopy was performed to observe the specific platelet granules using a microchip (K-kit; Material Analysis Technology Inc., Hsinchu, Taiwan), as described in a recent study. 16 The K-kit microchip provides a narrow chamber (300 μm long, 25 μm wide, and 2 μm high), which is appropriate for detecting isolated platelet granules. 16 In brief, the chamber was precoated with a poly-L-lysine solution for subsequent loading of isolated platelet granules.…”

“…16 The K-kit microchip provides a narrow chamber (300 μm long, 25 μm wide, and 2 μm high), which is appropriate for detecting isolated platelet granules. 16 In brief, the chamber was precoated with a poly-L-lysine solution for subsequent loading of isolated platelet granules. The samples were then fixed and incubated with anti-Pselectin mAb, followed by incubation with gold-conjugated goat antimouse IgG (6 nm).…”

Special Issue “Nature or Synthetic Compounds for Treating Arterial Thrombosis and Ischemic Stroke”

“…An interesting study conducted by Thu Trang et al [7] used a novel microchip (K-kit) as a specimen kit for the in situ imaging of human platelet granules in an aqueous solution using a transmission electron microscope (TEM). They suggested that this microchip technique might offer researchers the ability to observe the nanogranules of biological specimens in aqueous conditions in faster and better selections when applying a TEM.…”