Development of a direct point electron beam exposure system to investigate the biological functions of subcellular domains in a living biological cell. (April 2022)

• Record Type: Journal Article
• Authors: Tanaka, Asahi; Inami, Wataru; Suzuki, Yuko; Kawata, Yoshimasa
• Is Part Of: Micron. Volume 155(2022)
• Publication Date: 2022-04
• Subjects: Localized cell stimulation -- Living biological cell -- Electron beam -- Fluorescence imaging; Microscopy -- Periodicals; Electron Probe Microanalysis -- Periodicals; Microscopy -- Periodicals; Microscopie -- Périodiques; Microscopy; Periodicals; 502.82
• DOI: 10.1016/j.micron.2022.103214
• Languages: English
• ISSNs: 0968-4328

Abstract

Abstract: Electron microscopy studies have demonstrated that the diameter of a focused electron beam is small enough to probe or manipulate subcellular domains of a single biological cell. Here, we report the development of a direct point electron beam irradiation system to investigate the biological functions of subcellular domains in a living cell. Subcellular structures of a single living cell cultured on a thin film can be selectively irradiated by the point electron beam generated by our system. We have demonstrated controlled beam positioning capability to selectively irradiate 500 nm size structure with a point electron beam. We determined beam irradiation parameters that did not cause irreversible plasma membrane perforation after beam exposure and the irradiation caused intracellular Ca 2+ elevation in an irradiated neuronal cell. Since the neuronal cell express fine subcellular structures such as neurites, we tried to position a beam on the structure and observed a Ca 2+ wave originated from the intended point, which showed that our system had enough selectivity to target a subcellular structure. Point electron beam exposure is expected to be employed for various cellular stimulation protocols, and this enables the investigation of the biological functions of subcellular domains. Highlights: Localized cellular stimulation method using focused electron beam was developed. 2D Gaussian fitting was employed to achieve accurate beam positioning. Targeted beam positioning onto a neuronal subcellular structure was demonstrated. Stimulation that excites intracellular Ca 2+ elevation without irreversible plasma membrane perforation was achieved.