Abstract
We developed a high-resolution fluorescence microscope in which fluorescent materials are directly excited using a focused electron beam. Electron beam excitation enables detailed observations on the nanometer scale. Real-time live-cell observation is also possible using a thin film to separate the environment under study from the vacuum region required for electron beam propagation. In this study, we demonstrated observation of cellular components by autofluorescence excited with a focused electron beam and performed dynamic observations of intracellular granules. Since autofluorescence is associated with endogenous substances in cells, this microscope can also be used to investigate the intrinsic properties of organelles.
Highlights
In biological cells, cellular functions emerge as a result of localization and dynamic interaction of molecules
We developed a high-resolution fluorescence microscope in which fluorescent materials are directly excited using a focused electron beam
Real-time live-cell observation is possible using a thin film to separate the environment under study from the vacuum region required for electron beam propagation
Summary
Cellular functions emerge as a result of localization and dynamic interaction of molecules. To investigate the distribution and movement of such molecules in real time, several high-resolution microscopy techniques using light or electrons have been developed. Autofluorescence analysis, in which cellular molecules are optically excited without the need for a fluorescent dye, is useful for investigating the chemical composition of organelles. We have previously reported the development of a very-highresolution direct electron-beam excitation assisted (D-EXA) optical microscope for dynamic observation of living cells [16]. Fluorescent materials attached as labels to specific cellular molecules or endogenous fluorophores are directly irradiated with an electron beam, and the resulting CL is detected. Such electron beam excitation enables achievement of nanometerscale resolution, beyond the diffraction limit of light. Since some cellular substances emit CL, the D-EXA microscope enables label-free imaging in addition to qualitative chemical analysis using the CL spectrum
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