Dynamic and high-resolution live cell imaging by direct electron beam excitation

Nawa, Yasunori; Inami, Wataru; Chiba, Akito; Ono, Akira; Miyakawa, Atsuo; Kawata, Yoshimasa; Lin, Shir‐Kuan; Terakawa, Susumu

doi:10.1364/oe.20.005629

Cited by 42 publications

(36 citation statements)

References 20 publications

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“…However, to achieve the maximal resolution of~10 nm, a considerable electron dose and heavy metal staining was needed on account of the usage of the backscatter detector in SEM, and the depth range was also limited. The electron beam can also be used to generate small light spots illuminating cells (Nawa et al, 2012).…”

Section: Electron Microscopy In Liquidmentioning

confidence: 99%

Liquid Scanning Transmission Electron Microscopy: Imaging Protein Complexes in their Native Environment in Whole Eukaryotic Cells

Peckys

Jonge

2014

Microsc Microanal

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Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

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Section: Electron Microscopy In Liquidmentioning

confidence: 99%

Liquid Scanning Transmission Electron Microscopy: Imaging Protein Complexes in their Native Environment in Whole Eukaryotic Cells

Peckys

Jonge

2014

Microsc Microanal

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“…[40] The spot size used in the D-EXA microscope was calculated to a few tens of nanometers after penetrating the SiN membrane with a thickness of 50 nm at an acceleration voltage of 5 kV, [15] which is larger than that in STEM. [13] In the case of a spot size with a diameter of 30 nm in the D-EXA microscope, the electron beam was continuously irradiated in the same area with a density of about 80 electrons nm À2 (dwell time: 19 ms, probe current: 478 pA), which is two orders of magnitude below the amount used in STEM with a spot size of less than 1 nm in diameter.…”

Section: à2mentioning

confidence: 99%

Multi‐Color Imaging of Fluorescent Nanodiamonds in Living HeLa Cells Using Direct Electron‐Beam Excitation

et al. 2014

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Multi-color, high spatial resolution imaging of fluorescent nanodiamonds (FNDs) in living HeLa cells has been performed with a direct electron-beam excitation-assisted fluorescence (D-EXA) microscope. In this technique, fluorescent materials are directly excited with a focused electron beam and the resulting cathodoluminescence (CL) is detected with nanoscale resolution. Green- and red-light-emitting FNDs were employed for two-color imaging, which were observed simultaneously in the cells with high spatial resolution. This technique could be applied generally for multi-color immunostaining to reveal various cell functions.

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“…We previously developed electron beam excitation-assisted (EXA) optical (Inami et al 2010) and direct electron beam excitation-assisted (D-EXA) optical (Nawa et al 2012) microscopies. These techniques use a point light source as a probe to obtain resolutions above the diffraction limit.…”

Section: Introductionmentioning

confidence: 99%

Cell culture on hydrophilicity-controlled silicon nitride surfaces

Masuda

Inami

Miyakawa

et al. 2015

World J Microbiol Biotechnol

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Cell culture on silicon nitride membranes is required for atmospheric scanning electron microscopy, electron beam excitation assisted optical microscopy, and various biological sensors. Cell adhesion to silicon nitride membranes is typically weak, and cell proliferation is limited. We increased the adhesion force and proliferation of cultured HeLa cells by controlling the surface hydrophilicity of silicon nitride membranes. We covalently coupled carboxyl groups on silicon nitride membranes, and measured the contact angles of water droplets on the surfaces to evaluate the hydrophilicity. We cultured HeLa cells on the coated membranes and evaluated stretch of the cell. Cell migration and confluence were observed on the coated silicon nitride films. We also demonstrated preliminary observation result with direct electron beam excitation-assisted optical microscope.

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Dynamic and high-resolution live cell imaging by direct electron beam excitation

Cited by 42 publications

References 20 publications

Liquid Scanning Transmission Electron Microscopy: Imaging Protein Complexes in their Native Environment in Whole Eukaryotic Cells

Liquid Scanning Transmission Electron Microscopy: Imaging Protein Complexes in their Native Environment in Whole Eukaryotic Cells

Multi‐Color Imaging of Fluorescent Nanodiamonds in Living HeLa Cells Using Direct Electron‐Beam Excitation

Cell culture on hydrophilicity-controlled silicon nitride surfaces

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