우리는 ESRF에서 자신의 도움을 파보 누르 버그만, 마이클 라우먼, 그리고 세바스찬 Schmelzle 감사드립니다. 이 작품은 빔 시간의 배분을 통해 유럽 싱크로 트론 방사선 시설 사업 SC - 2127에 의해 지원되었다.

이 연구에 사용된 동물 parthenogenetic oribatid 마이트 Archegozetes longisetosus (Acari, Oribatida)의 표본은 우리의 연구실 문화에서 찍은. 문화가 20-23에서 지속적인 어둠 속에서, 파리 / 플라스틱 항아리에 숯 믹스 (9시 1분)의 석고의 성장 ° C 공기 습도 약 90 %를. 샘플 준비 <ol><li> 표본은 좋은 브러시로 청소하고 24 시간 동안 80 % 에탄올, 35 % 포름 알데히드 100 % 초산의 6시 3분 1초 혼합 배치, 문화에서 찍은. </li><li> 그 후, 표본은 각 농도 3 변경하고, 단계 사이에 10 분있는 등급 에탄올 70 % 시리즈, 75 %, 80 %, 85 %, 90 %, 95%, 100 %에 탈수있었습니다. </li><li> 마지막으로 샘플은 CO 2의 건조 신선한 100 % 에탄올 하룻밤과 임계점 (CPD 020, Balzers)에 배치했다. 말린 표본는 플라스틱 핀이의 끝 (; 직경 3.0 mm 길이 1.2 ㎝)에 첨부했습니다. </li></ol> 싱크로 트론 X - 선 tomography X - 선 tomography는 beamline ID19 (ESRF, 그르노블, 프랑스,​​ 실험 SC - 2127)에서 수행되었다. <ol><li> 샘플은 샘플 홀더에 장착하고 빔의 중앙 위치로 조정되었습니다. </li><li> 샘플은 20.5 케빈의 에너지를 측정했다. radiographs은 14 비트 다이나믹 레인지로 냉각 CCD (ESRF 프렐론 카메라), 2,048 × 2,048 픽셀과 0.7 μm의의 효과적인 픽셀 크기로 기록되었다. 1500 계획은 각 투영 0.35 s의 노출 시간과 180 ° 샘플 회전을 통해 기록했다. 검출기 - 투 - 샘플 거리가 20mm되었습니다. </li></ol> 샘플과 검출기 사이에 일정한 거리를 사용하면 (샘플은 검출기의 바로 앞에 위치 흡수 이미징에 충분한 대비를 생성 낮은 X - 선 감쇠 계수 (Cloetens, 외. 1996)과 재료의 차등 이미징을 가능하게 ). 대부분의 생물 학적 문제는 낮은 흡수 및 / 또는 원자 번호에만 작은 차이 (베츠, 외. 2007)과 재료의 구성 단계 개체입니다. 그러나, 위상 강화 tomography은 단일 X - 선 빔 높은 공간적 일관성이 필요합니다. 따라서, 싱크로 트론 방사는 더 나은 측정 이러한 종류의 데스크탑 스캐너보다 적합합니다. 데이터 분석 <ol><li> 그 결과 2D radiographs은 필터링된 다시 프로젝션 알고리즘 (Cloetens, 외., 1997)와 3D voxel 데이터 (8 비트 회색 값)으로 변형되었다 </li><li> voxel 데이터는 소프트웨어 VGStudio 최대 1.2.1로 분석했다. (볼륨 그래픽, 하이 델베르크, 독일). </li><li> 배경에서 그레이 - 값이 3D - 시각화에 대한 히스토그램에서 제거되었습니다. </li><li> 사전 정의된 카메라 경로는 회전 애니메이션과 애니메이션 클리핑 비행기를 생성하는 데 사용되었습니다. </li><li> 사용자 정의 카메라 경로 A.의 소화 시스템에 따라 생성되었습니다 longisetosus. </li></ol>

<ol>
	<li>Betz, O., Wegst, U., Weide, D., Heethoff, M., Helfen, L., Lee, W. -. K., Cloetens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+applications+of+synchrotron+X-ray+phase-contrast+microtomography+in+biological+morphology+and+biomaterial+science.+I.+General+aspects+of+the+technique+and+its+advantages+in+the+analysis+of+millimetre-sized+arthropod+structure.">Imaging applications of synchrotron X-ray phase-contrast microtomography in biological morphology and biomaterial science. I. General aspects of the technique and its advantages in the analysis of millimetre-sized arthropod structure.</a> J. Microscopy. 22, 51-71 (2007).</li><li>Cloetens, P., Barrett, R., Baruchel, J., Guigay, J. P., Schlenker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+objects+in+synchrotron+radiation+hard+X-ray+imaging.">Phase objects in synchrotron radiation hard X-ray imaging.</a> J. Phys. D: Appl. Phys. 29, 133-146 (1996).</li><li>Cloetens, P., Pateyron-Salome, M., Buffiere, J. Y., Peix, G., Baruchel, J., Peyrin, V., Schlenker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+in+microstructure+and+damage+in+materials+by+phase+sensitive+radiography+and+tomography.">Observation in microstructure and damage in materials by phase sensitive radiography and tomography.</a> J. Apll. Phys. 81, 5878-5886 (1997).</li><li>Clotens, P., Ludwig, W., Baruchel, J., van Dyck, D., van Landyut, J., Guigay, J. P., Schlenker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holotomography:+quantitative+phase+tomography+with+micrometer+resolution+using+hard+synchrotron+radiation+X-rays.">Holotomography: quantitative phase tomography with micrometer resolution using hard synchrotron radiation X-rays.</a> Appl. Phys. Lett. 75, 2912-2914 (1999).</li><li>Heethoff, M., Cloetens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+aynchrotron+X-ray+phase+contrast+tomography+and+holotomography+for+non-invasive+investigations+of+the+internal+anatomy+of+mites.">A Comparison of aynchrotron X-ray phase contrast tomography and holotomography for non-invasive investigations of the internal anatomy of mites.</a> Soil Organisms. , (2008).</li></ol>

The authors have nothing to disclose.

이 프레 젠 테이션에서, 우리는 chelicerate 마이크로 arthropod의 내부 해부의 3D - 시각화에 초점을 맞추었습니다. 싱크로 트론 X - 선 측정은 샘플의 크기에 따라, 0.3μm로의 픽셀 해상도를 허용합니다. 여기, 우리는 픽셀 해상도 0.7μm 데이터를 표시합니다. 일반적으로, 싱크로 트론 X - 선 tomography 낮은 X - 선 감쇠와 함께 작은 생물 학적 물질 (또는 조직)를 분석하는 데 유용할 수 있습니다. 픽셀 해상도는 거의 전통적인...

non-invasive 3d-visualization with sub-micron resolution using synchrotron-x-ray-tomography

리틀은 1mm 아래의 신체 크기와 많은 마이크로 arthropods의 내부 조직에 대한 알려져있다. 에 대한 이유는 작은 크기와 어려운 클래식 조직학의 프로토콜을 사용할 수 있습니다 하드 표피입니다. 또한, histological sectioning는 샘플을 파괴하기 때문에 독특한 소재에 대해 사용할 수 없습니다. 따라서 비파괴 방법 sectioning의 필요없이 작은 샘플 안에서 볼 수 있습니다하는 것이 바람직하다. 우리는 비 invasively 0.7μm의 픽셀 해상도와 3D 단층 데이터 세트를 생성 그르노블 (프랑스)의 유럽 싱크로 트론 방사선 시설 (ESRF)에 싱크로 트론 X - 선 tomography를 사용했습니다. 볼륨 렌더링 소프트웨어를 사용, 이것은 우리 histological sectioning 제작한 공예품없이 자연 상태에서 내부 조직을 재구성 할 수 있습니다. 이 날짜는 숨겨진 신체 부위의 구조를 이해하고 예제를 통해 전체 기관의 시스템이나 조직에 따라 정량 형태, 랜드마크, 또는 애니메이션 영​​화의 시각화를 위해 사용할 수 있습니다.

Watch this Scientific Journal Video about Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography at JoVE.com

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

우리는 비 invasively 0.7μm의 픽셀 해상도와 3D 단층 데이터 세트를 생산하는 유럽 싱크로 트론 방사선 시설 (ESRF)에 싱크로 트론 X - 선 tomography를 사용했습니다. 볼륨 렌더링 소프트웨어를 사용하여이 histological sectioning 제작한 공예품없이 자연 상태에서 내부 구조의 재건 수 있습니다.

싱크로 트론 - X - 레이 - tomography를 사용하여 서브 마이크론 해상도로 비침습 3D - 시각화

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

non-invasive-3d-visualization-with-sub-micron-resolution-using

Research

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Biology

13.1K 조회수.  University of Tubingen. 우리는 비 invasively 0.7μm의 픽셀 해상도와 3D 단층 데이터 세트를 생산하는 유럽 싱크로 트론 방사선 시설 (ESRF)에 싱크로 트론 X - 선 tomography를 사용했습니다. 볼륨 렌더링 소프트웨어를 사용하여이 histological sectioning 제작한 공예품없이 자연 상태에서 내부 구조의 재건 수 있습니다.

비디오: Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

고해상도 3D 영상의 전 VIVO</em마이크로 중부 표준시로> 생물 학적 샘플

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

연구

생체공학

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

멀티 앵글 조명 및 픽셀 슈퍼 해상도를 채용 Lensfree 온칩 단층 현미경

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data acquisition times with high spatial resolution. In the electronics industry there is serious interest in performing failure analysis on 3D microelectronic packages, many which contain multiple levels of high-density interconnections. Often in tomography there is a trade-off between image resolution and the volume of a sample that can be imaged. This inverse relationship limits the usefulness of conventional computed tomography (CT) systems since a microelectronic package is often large in cross sectional area 100-3,600 mm2, but has important features on the micron scale. The micro-tomography beamline at the Advanced Light Source (ALS), in Berkeley, CA USA, has a setup which is adaptable and can be tailored to a sample&#39;s properties, i.e., density, thickness, etc., with a maximum allowable cross-section of 36 x 36 mm. This setup also has the option of being either monochromatic in the energy range ~7-43 keV or operating with maximum flux in white light mode using a polychromatic beam. Presented here are details of the experimental steps taken to image an entire 16 x 16 mm system within a package, in order to obtain 3D images of the system with a spatial resolution of 8.7 &#181;m all within a scan time of less than 3 min. Also shown are results from packages scanned in different orientations and a sectioned package for higher resolution imaging. In contrast a conventional CT system would take hours to record data with potentially poorer resolution. Indeed, the ratio of field-of-view to throughput time is much higher when using the synchrotron radiation tomography setup. The description below of the experimental setup can be implemented and adapted for use with many other multi-materials.

For this study synchrotron radiation micro-tomography, a non-destructive three-dimensional imaging technique, is employed to investigate an entire microelectronic package with a cross-sectional area of 16 x 16 mm. Due to the synchrotron&#39;s high flux and brightness the sample was imaged in just 3 min&#160;with an 8.7 &#181;m spatial resolution.

멀티 스케일 3 차원 마이크로 전자 패키지를 조사하는 방사광 Microtomography 사용

Synchrotron radiation micro-tomography (SR&#181;T) is a non-destructive three-dimensional (3D) imaging technique that offers high flux for fast data ...

공학

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

싱크로 빠른 단층 촬영을 사용하여 탄산염의 반응의 동적 기공 규모의 저수지 조건 이미징

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

Visualization of Failure and the Associated Grain-Scale Mechanical Behavior of Granular Soils under Shear using Synchrotron X-Ray Micro-Tomography

The rapid development of X-ray imaging techniques with image processing and analysis skills has enabled the acquisition of CT images of granular soils with high-spatial resolutions. Based on such CT images, grain-scale mechanical behavior such as particle kinematics (i.e., particle translations and particle rotations), strain localization and inter-particle contact evolution of granular soils can be quantitatively investigated. However, this is inaccessible using conventional experimental methods. This study demonstrates the exploration of the grain-scale mechanical behavior of a granular soil sample under triaxial compression using synchrotron X-ray micro-tomography (&#956;CT). With this method, a specially fabricated miniature loading apparatus is used to apply confining and axial stresses to the sample during the triaxial test. The apparatus is fitted into a synchrotron X-ray tomography setup so that high-spatial resolution CT images of the sample can be collected at different loading stages of the test without any disturbance to the sample. With the capability of extracting information at the macro scale (e.g., sample boundary stresses and strains from the triaxial apparatus setup) and the grain scale (e.g., grain movements and contact interactions from the CT images), this procedure provides an effective methodology to investigate the multi-scale mechanics of granular soils.

The protocol describes procedures to acquire high-spatial resolution computed tomography (CT) images of a granular soil during triaxial compression, and to apply image processing techniques to these CT images to explore the grain-scale mechanical behavior of the soil under loading.

싱크로트론 X선 마이크로 단층 촬영을 사용하여 전단 하에서 세분화된 토양의 고장 및 관련 곡물 스케일 기계적 거동 시각화

The rapid development of X-ray imaging techniques with image processing and analysis skills has enabled the acquisition of CT images of granular soils ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

마이크로 컴퓨팅 단층 촬영을 사용하여 드로소필라 멜라노가스터의 전체 동물 이미징

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

발생학

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.

A protocol for 3D visualization of microscopic tissue structures by using an X-ray specific staining method designed for X-ray computed tomography is presented.

X선 특이적 염색 방법 및 나노스코픽 컴퓨터 단층 촬영을 사용한 연조직 시료의 3D 이미징

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

공장 Vasculature의 3 차원 구조 및 기능을 시각화하기 위해 높은 해상도로 계산 된 단층 촬영을 사용하여

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

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A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Cryo Soft X선 단층 촬영에 의한 세포의 3D Cartographic 설명

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 &#181;m.

싱크로 트론 - X - 레이 - tomography를 사용하여 서브 마이크론 해상도로 비침습 3D - 시각화

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