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Current smartphones equipped with high-sensitivity and high-resolution sensors in the camera can respond to the needs of low-light imaging, streaming acquisition, targets of various scales, etc. Therefore, a smartphone has great potential as an imaging device even in the scientific field and has already been introduced into biomolecular imaging using fluorescence tags. However, owing to the necessity of an excitation light source, fluorescence methods impair its mobility. Bioluminescence does not require illumination; therefore, imaging with a smartphone camera is compact and requires minimal devices, thus making it suitable for personal and portable imaging devices. Here, we report smartphone-based methods to observe biological targets in various scales using bioluminescence. In particular, we demonstrate, for the first time, that bioluminescence can be observed in an organelle in a single living cell using a smartphone camera by attaching a detachable objective lens. Through capturing color changes with the camera, changes in the amount of target molecules was detected using bioluminescent indicators. The combination of bioluminescence and a mobile phone makes possible a compact imaging system without an external light source and expands the potential of portable devices.
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Técnicas Biossensoriais , Organelas , Smartphone , Animais , Telefone Celular , Iluminação , CamundongosRESUMO
High-voltage transmission electron microscopes (HVTEMs), which can visualize internal structures of micron thick samples, intrinsically have large instrument sizes because of the static voltage isolation. In this Letter, we develop a compact HVTEM, employing a linear accelerator, a subpicosecond beam chopper, and a linear decelerator. 100 kV electrons initially accelerated by a static field are accelerated at radio frequency (rf) up to 500 kV, transmitting through the sample and finally rf decelerated down to 200 kV to be imaged through a 200 kV energy filter. 500 kV imaging, as well as subnanometer resolution at 200 kV, have been demonstrated.
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Cyanobacteria are photosynthetic organisms responsible for â¼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.
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Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/ultraestrutura , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Synechococcus/ultraestrutura , Synechococcus/virologia , Montagem de Vírus , Organismos Aquáticos/citologia , Organismos Aquáticos/ultraestrutura , Organismos Aquáticos/virologia , Modelos Biológicos , Synechococcus/citologiaRESUMO
Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads.
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Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Animais , Células Cultivadas , Elétrons , Fluorescência , Fótons , PotoroidaeRESUMO
Cryo-electron tomography of frozen hydrated cells has provided cell biologists with an indispensable tool for delineating three-dimensional arrangements of cellular ultrastructure. To avoid the damage induced by electron irradiation, images of frozen hydrated biological specimens are generally acquired under low-dose conditions, resulting in weakly contrasted images that are difficult to interpret, and in which ultrastructural details remain ambiguous. Zernike phase contrast transmission electron microscopy can improve contrast, and can also fix a fatal problem related to the inherent low contrast of conventional electron microscopy, namely, image modulation due to the unavoidable setting of deep defocus. In this study, we applied cryo-electron tomography enhanced with a Zernike phase plate, which avoids image modulation by allowing in-focus setting. The Zernike phase contrast cryo-electron tomography has a potential to suppress grainy background generation. Due to the smoother background in comparison with defocus phase contrast cryo-electron tomography, Zernike phase contrast cryo-electron tomography could yield higher visibility for particulate or filamentous ultrastructure inside the cells, and allowed us to clearly recognize membrane protein structures.
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Membrana Celular/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica/métodos , Dipodomys , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador , Proteínas de Membrana/ultraestrutura , Microscopia de Contraste de Fase/métodos , Análise de Célula Única/métodosRESUMO
This Commentary describes the June 2022 installment of the "Editors' Roundup" feature. The Editors' Roundup is a multi-author collective description of recently published biophysical research contributed by editorial board members of different journals with a bonafide interest in publishing biophysical content. This Commentary contains a series of personal recommendations for articles appearing in the following journals, Biophysics and Physicobiology, European Biophysics Journal, Cell Biochemistry and Biophysics, and Biophysical Reviews.
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Vibrio alginolyticus use flagella to swim. A flagellum consists of a filament, hook and basal body. The basal body is made up of a rod and several ring structures. This study investigates the structure of the T ring which is a unique component of the V. alginolyticus sodium ion-driven flagellar basal body. Using Zernike phase contrast (ZPC) cryo-electron tomography, we compared the 3D structures of purified hook-basal bodies (HBB) from a wild-type strain (KK148) and a deletion mutant lacking MotX and MotY (TH3), which are thought to form the T ring. ZPC images of HBBs had highly improved signal-to-noise ratio compared to conventional phase contrast images. We observed the outline of the HBBs from strains KK148 and TH3, and the TH3 mutant was missing its T ring. In the wild-type strain, the T ring was beneath the LP ring and seemed to form a ring shape with diameter of 32 nm.
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Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Flagelos/ultraestrutura , Microscopia de Contraste de Fase/métodos , Vibrio/citologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Deleção de Genes , Processamento de Imagem Assistida por Computador , Proteínas de Membrana/genética , Vibrio/genéticaRESUMO
The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel responsive to various stimuli including cell swelling, warm temperatures (27-35 degrees C), and chemical compounds such as phorbol ester derivatives. Here we report the three-dimensional structure of full-length rat TRPV4 purified from baculovirus-infected Sf9 cells. Hexahistidine-tagged rat TRPV4 (His-rTRPV4) was solubilized with detergent and purified through affinity chromatography and size-exclusion chromatography. Chemical cross-linking analysis revealed that detergent-solubilized His-rTRPV4 was a tetramer. The 3.5-nm structure of rat TRPV4 was determined by cryoelectron microscopy using single-particle reconstruction from Zernike phase-contrast images. The overall structure comprises two distinct regions; a larger dense component, likely corresponding to the cytoplasmic N- and C-terminal regions, and a smaller component corresponding to the transmembrane region.
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Microscopia Crioeletrônica/métodos , Microscopia de Contraste de Fase/métodos , Canais de Cátion TRPV/química , Animais , Cálcio/química , Linhagem Celular , Cromatografia de Afinidade/métodos , Reagentes de Ligações Cruzadas/química , Detergentes/farmacologia , Processamento de Imagem Assistida por Computador , Insetos , Microscopia Eletrônica de Transmissão/métodos , Conformação Molecular , Conformação Proteica , RatosRESUMO
It has been six decades since the concept of phase-plate electron microscopy was first reported by Boersch, but an experimental report on a phase plate with a theoretically rational performance has only recently been released by a group including the present author. Currently, many laboratories around the world are attempting to develop a wide range of phase plates to enhance the capabilities of transmission electron microscopy. They are reporting not only advantages of their own developments but also a fundamental problem inherent to electron beam devices, namely charging, i.e. the accumulation of electrostatic charge. In this report, we review the 60-year history of phase-plate development, with a particular focus on the fundamental issue of phase-plate charging. Next, we review biological applications of qualified phase plates, which have been successful in avoiding charging to some extent. Finally, we compare and discuss electron microscopic images, taken with or without phase plates, of biological targets such as proteins (GroEL and TRPV4), protein complexes (flagellar motor), viruses (T4 phage, ε-15 phage and herpes simplex virus), bacterial (cyanobacteria) and mammalian (PtK2) cells.
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Microscopia Eletrônica de Transmissão , Proteínas/ultraestrutura , Células/ultraestrutura , Flagelos/ultraestrutura , Microscopia Eletrônica de Transmissão/instrumentação , Microscopia Eletrônica de Transmissão/métodos , Eletricidade Estática , Vírus/ultraestruturaRESUMO
Cryo-tomography in the electron microscope is unique in its ability to provide high-resolution, three-dimensional structural information about cells, organelles and macromolecules in a nearly native, frozen-hydrated state. However, the phase-contrast imaging method used in conventional cryo-electron tomography fails to faithfully represent the full range of structural features in such specimens. Only certain features are recorded with adequate contrast, and overall contrast is low. The recently developed Zernike phase contrast method has the potential to solve this problem, and here we apply it for the first time to cryo-electron tomography. The new method has uniform transfer characteristics for a wide range of spatial frequencies, leading to improved overall signal-to-noise ratio and raising the prospects of higher resolution and quantitative representation of specimen densities in the reconstructed tomograms.
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Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodosRESUMO
In this second instalment of the Biophysical Reviews' Meet the Editors Series we hear the story of Prof. Kuniaki Nagayama, one of the five Executive Editors of Biophysical Reviews.
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In order to acquire phase-contrast images with adequate contrast, conventional TEM requires large amount of defocus. Increasing the defocus improves the low-frequency components but attenuates the high-frequency ones. On the other hand, Zernike phase-contrast TEM (ZPC-TEM) can recover low-frequency components without losing the high-frequency ones under in-focus conditions. ZPC-TEM however, has another problem, especially in imaging of complex biological specimens such as cells and tissues; strong halos appear around specimen structures, and these halos hinder the interpretation of images. Due to this problem, the application of ZPC-TEM has been restricted to imaging of smaller particles. In order to improve the halo appearance, we fabricated a new quarter-wave thin film phase-plate with a smaller central hole and tested it on vitreous biological specimens. ZPC-TEM with the new plate could successfully visualize, in in-focus images, the intracellular fine features of cultured cells and brain tissues. This result indicates that reduction of the central hole diameter makes ZPC-TEM applicable on size scales ranging from protein particles to tissue sections. The application of ZPC-TEM to vitreous biological specimens will be a powerful method to advance the new field of imaging science for ultrastructures in close-to-physiological state.
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Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Contraste de Fase/métodos , Animais , Encéfalo/ultraestrutura , Células Cultivadas , Técnicas In Vitro , Masculino , CamundongosRESUMO
A number of practical issues must be addressed when using thin carbon films as quarter-wave plates for Zernike phase-contrast electron microscopy. We describe, for example, how we meet the more stringent requirements that must be satisfied for beam alignment in this imaging mode. In addition we address the concern that one might have regarding the loss of some of the scattered electrons as they pass through such a phase plate. We show that two easily measured parameters, (1) the low-resolution image contrast produced in cryo-EM images of tobacco mosaic virus particles and (2) the fall-off of the envelope function at high resolution, can be used to quantitatively compare the data quality for Zernike phase-contrast images and for defocused bright-field images. We describe how we prepare carbon-film phase plates that are initially free of charging or other effects that degrade image quality. We emphasize, however, that even though the buildup of hydrocarbon contamination can be avoided by heating the phase plates during use, their performance nevertheless deteriorates over the time scale of days to weeks, thus requiring their frequent replacement in order to maintain optimal performance.
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Microscopia Eletrônica de Transmissão/instrumentação , Bactérias/ultraestrutura , Carbono/química , Microscopia Eletrônica de Transmissão/normasRESUMO
Phase contrast electron microscopy utilizing phase plates has been considered suitable for high-contrast observation of weak phase objects. This novel technique was newly applied to histochemically stained strong phase objects of osmificated biological specimens. Sections of various thicknesses, specifically stained for the Golgi apparatus by the ZIO technique using the heavy metals Zn and Os, were observed with a phase contrast electron microscope in Zernike and Hilbert imaging modes. Quantitative analysis of image contrast in real space and the power spectrum in Fourier space showed a high-contrast gain even for strong phase objects. This result clearly indicates that phase contrast electron microscopy can be effectively used not only for weak phase objects but also for strong phase objects in biology.
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Complexo de Golgi/ultraestrutura , Fígado/ultraestrutura , Microscopia Eletrônica/métodos , Microscopia de Contraste de Fase/métodos , Inclusão do Tecido/métodos , Animais , Masculino , Microscopia Eletrônica/instrumentação , Microscopia de Contraste de Fase/instrumentação , Plásticos , Ratos , Ratos WistarRESUMO
We present the first application of Zernike phase-contrast transmission electron microscopy to single-particle 3D reconstruction of a protein, using GroEL chaperonin as the test specimen. We evaluated the performance of the technique by comparing 3D models derived from Zernike phase contrast imaging, with models from conventional underfocus phase contrast imaging. The same resolution, about 12A, was achieved by both imaging methods. The reconstruction based on Zernike phase contrast data required about 30% fewer particles. The advantages and prospects of each technique are discussed.
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Microscopia Eletrônica de Transmissão/métodos , Microscopia de Contraste de Fase/métodos , Conformação Proteica , Chaperonina 60/químicaRESUMO
The ultrastructure of the frozen-hydrated influenza A virus was examined by Zernike phase contrast electron microscopy. Using this new microscopy, not only lipid bilayers but also individual glycoprotein spikes on viral envelopes were clearly resolved with high contrast in micrographs taken in focus. In addition to spherical and elongated virions, three other classes of virions were distinguished on the basis of the features of their viral envelope: virions with a complete matrix layer, which were the most predominant, virions with a partial matrix layer, and virions with no matrix layer under the lipid bilayer. About 450 glycoprotein spikes were present in an average-sized spherical virion. Eight ribonucleoprotein complexes, that is, a central one surrounded by seven others, were distinguished in one viral particle. Thus, Zernike phase contrast electron microscopy is a powerful tool for resolving the ultrastructure of viruses, because it enables high-contrast images of ice-embedded particles free of contrast transfer function artifacts that can be a problem in conventional cryo-electron microscopy.
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Gelo , Vírus da Influenza A/ultraestrutura , Microscopia de Contraste de Fase/métodos , Microscopia Crioeletrônica , Glicoproteínas , Vírus da Influenza A/química , Bicamadas Lipídicas , Proteínas do Envelope Viral , VírionRESUMO
We previously reported that transferrin (Tf)-modified liposomes (Tf-L) additionally modified with a cholesterylated pH-sensitive fusogenic peptide (Chol-GALA) can release an encapsulated aqueous phase marker to cytosol via endosomal membrane fusion. However, further obstacles need to be overcome to bring the Tf-L to the level of a viral-like gene delivery system. In this study, we developed a novel packaging method to encapsulate condensed plasmid DNA into PEgylated Tf-L (Tf-PEG-L) to form a core-shell-type nanoparticle. The most difficult challenge was to provide a mechanism of escape for the condensed core from endosome to cytosol in the presence of polyethylene glycol (PEG). We hypothesized that a membrane-introduced Chol-GALA and a PEgylated GALA would interact synergistically to induce membrane fusion between liposome and endosome. By simultaneously incorporating Chol-GALA into the membrane of Tf-PEG-L and GALA at tips of PEG chains, a condensed core was released into cytosol, and transfection activity increased 100-fold. We concluded that topological control was responsible for the synergistic effect of GALA derivatives introduced on Tf-PEG-L.
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Endossomos , Técnicas de Transferência de Genes , Fusão de Membrana , Nanopartículas/química , Peptídeos/química , Virossomos/química , Colesterol/química , Endossomos/química , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Células K562 , Peptídeos/metabolismo , Polietilenoglicóis/química , Transferrina/química , Transferrina/metabolismo , Virossomos/metabolismoRESUMO
Oligoarginine conjugates are highly efficient vectors for the delivery of plasmid DNA into cells. Decaarginine-conjugated lipid (Arg10-PEG-lipid) was synthesized and the effects of Arg10-PEG-lipid concentration at a fixed DNA concentration on transfection efficiency and the structure of the complexes were studied below and above critical micelle concentration (CMC), and at the lipid nitrogen/DNA phosphate (N/P) ratio corresponding to transfection, respectively. Arg10-PEG-lipid at the concentration below CMC showed stronger interaction with DNA by fluorescence intensity distribution analysis, and significantly higher luciferase and green fluorescent protein expression than that above CMC. A phase-contrast cryo-transmission electron microscope (cryo-TEM) experiment showed that the morphology of the complexes depended on the N/P ratio. At a low N/P ratio corresponding to that in transfection at a lipid concentration below CMC, a net-like structure developed in which plasmid DNA was involved. A further increase in the N/P ratio, a large fibrous nanostructure of complexes, was also observed. Without DNA, these structures were not obtained. The cellular uptake mechanism of complexes using flow cytometry with inhibitors suggested that complexes with two different morphologies showed similar cellular uptake and uptake mechanism, macropinocytosis. Differences in transfection efficiency of the complexes may be explained by a large fibrous nanostructure inhibiting the cellular internalization of complexes or the release of DNA from macropinosomes into cytoplasm. Arg10-PEG-lipid/DNA complexes formed a favorable nanostructure for gene delivery, depending on the N/P ratio in water.
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Arginina/química , DNA/química , Lipídeos/química , Nanoestruturas , Polietilenoglicóis/química , Transfecção , Fluorescência , Células HeLa , Humanos , Microscopia EletrônicaRESUMO
The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.