S-Nitrosoglutathione-induced mouse thymocyte apoptosis studied by fluorescence near-field scanning optical microscopy

Abstract

This study is an attempt to deeply understand the mechanisms ensuring self-tolerance of T cells via clonal deletion of thymocytes and exploring T lymophocyte homeostasis by observing the apoptosis of single mouse thymocyte induced by S-nitrosoglutathione (GSNO, a nitric oxide donor) using fluorescence near-field scanning optical microscopy (NSOM) in illumination mode. The GSNO-induced thymocytes were stained with propidium iodide containing 0.01% Triton X-100 and excited with light of 488 nm and the emitting fluorescence at 525 nm. According to the NSOM fluorescence image and the simultaneously obtained topography image, the feature of mouse thymocyte apoptosis was characterized by scattering pattern of the fluorescence spots with the size 0.2–2.1 μm at the full width at half-maximum of fluorescence intensity 78–80 kHz in the GSNO-treated thymocyte nucleus. Whereas there is no fluorescence from the untreated thymocyte. The intensity of the fluorescence from the dexamethasone-treated thymocyte was much stronger than that from GSNO-induced thymocytes. Furthermore, the fluorescence distribution in the latter were concentrated in the nucleus. Those results also demonstrate the advantages of NSOM such as high spatial resolution and the topography of biology samples.