Immuno EM–OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM)

Maruyama, Yasushi; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Suga, Mitsuo; Sato, Chikara

doi:10.1016/j.jsb.2012.08.006

Cited by 56 publications

(66 citation statements)

References 42 publications

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“…On the other hand, ASEM allows the observation of samples in aqueous solution; thus, sample preparation time can be significantly shortened (Nishiyama et al, ). Because all processes are performed in aqueous solution, antigenicity is well preserved (Maruyama et al, ). Consequently, the throughput of observation can be significantly improved compared with that for conventional electron microscopy.…”

Section: Discussionmentioning

confidence: 99%

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

Akita

Tanaka

Murai

et al. 2013

Microscopy Res & Technique

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We examined CD133 distribution in a human hepatoblastoma cell line (HuH-6 clone 5). We directly observed the cultured cells on a pressure-resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co-localization of CD133 with F-actin. An antibody against CD133 decreased cell migration. Methyl-β-cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration.

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Section: Discussionmentioning

confidence: 99%

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

Akita

Tanaka

Murai

et al. 2013

Microscopy Res & Technique

Self Cite

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“…De Jonge and co-workers have established correlative fluid cell STEM and fluorescence microscopy as a powerful technique for studying various aspects of eukaryotic cells, such as locating epidermal growth factor receptors in COS7 cells 38 , exploring the correlation between cellular function and ultrastructure in yeast cells 39 , and investigating electron beam damage and cell viability in live COS7 cells 43 . Other electron microscopy techniques exist for imaging whole hydrated cells, such as environmental scanning electron microscopy (ESEM) 44 , and air SEM (ASEM) 45 . The cells in these techniques are typically chemically fixed and maintained in a vacuum, making live cell imaging impossible; however, important information about cell receptors can be obtained by incubating the cells with nanoparticles prior to fixation and imaging 44 .…”

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confidence: 99%

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

et al. 2015

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Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ,30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.B iomineralization is a widespread biological phenomenon occurring in living organisms ranging from single cells to complex multicellular organisms. Biomineralization of inorganic materials in single cell organisms is an ideal system for studying fundamental mechanisms of biomineralization 1 . Model systems range from prokaryotic organisms, such as magnetite formation in magnetotactic bacteria 2-4 , to eukaryotic organisms, such as silica biomineralization in diatoms 5,6 . Understanding biomineralization in these organisms in terms of crystal nucleation and growth, as well as the involvement of biological macromolecules and cellular processes, is of fundamental interest to scientists as similar principles can be used to develop synthetic nanomaterials.Magnetite biomineralization by magnetotactic bacteria is a topic of great interest in nanotechnology 7-12 , functional materials [11][12][13][14][15][16] , and astrobiology 17 . Magnetotactic bacteria take up soluble iron species that they use to biomineralize chains of magnetite nanocrystals, known as magnetosomes, in intracellular membrane vesicles 2 . The nanocrystals have nearly perfect mineral crystal structures with consistent species-specific morphologies, leading to well-defined magnetic properties 2,3,9,12,18,19 . As a result, magnetotactic bacteria are one of the best model systems for investigating the molecular mechanisms of biomineralization. Magnetite biomineralization in these bacteria is a complex process involving a number of steps including cellular uptake and reduction of ferric ions, complexation of the iron with membrane proteins, an...

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“…The ultimate future perspective with combined FM and scanning EM in liquid would be to correlate live-cell imaging directly with an ultrastructural snapshot, the recording of which will deliver a lethal dose to the system under study. Liquid CLEM can be conducted by quickly transferring the microfluidic chamber between microscopes 95 , but instrumentation for integrated liquid CLEM, to match the EM snapshot with FM acquisitions in time, is also emerging 34 .…”

Section: Future Outlook Probes and Live-cell Electron Microscopymentioning

confidence: 99%

“…In the atmospheric SEM (ASEM, or Jeol's ClairScope) 33,34 and similar systems 35 , the fluorescence microscope and SEM are also positioned in paraxial configuration, but with the SEM inverted. Only back-scattered electrons can be detected through a thin (50-to 100-nm) silicon nitride membrane 36,37 , which seals the SEM vacuum chamber (Fig.…”

mentioning

confidence: 99%

Correlated light and electron microscopy: ultrastructure lights up!

2015

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Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development-including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy-now paves the way for broad CLEM implementation in biology.

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Immuno EM–OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM)

Cited by 56 publications

References 42 publications

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

Correlated light and electron microscopy: ultrastructure lights up!

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