우리는 증명 원리 실험, 고정 프로토콜 시약과 격려를 공유 리차드 족쇄를 채우다을위한 PALM 현미경에 대한 접근 랄드 헤스와 에릭 Betzig 감사드립니다. 우리는 DNA 구조에 대한 마이클 데이비슨, 제랄딘 Seydoux, 스테판 Eimer, 루돌프 Leube, 키이스 Nehrke, 기독교 Frøk​​jr - 젠슨, 오드 에이다 - Nguema과 마크 Hammarlund 감사드립니다. 우리는 또한 Zeiss PAL-M, Zeiss Elyra P.1 PALM 현미경의 베타 버전에 대한 액세스를 제공하기 위해 칼 Zeiss 주식회사 감사드립니다.

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이 기사에 생산 및 무료 액세스 칼 Zeiss 주식회사가 후원합니다

여기 플라스틱, 전자 현미경을 사용하여 ultrastructure을 섹션에 형광 단백질을 현지화 및 이미지 형광 단백질을 유지하는 방법에 대해 설명합니다. 단백질은 나노 미터 해상도 PALM 현미경을 사용하여 회절 한계 이하로 현지화되었습니다. 특정 표본이 프로토콜을 적응하려면 다음 매개 변수 고려되어야합니다 : 형광, 정량화 및 정렬을. 형광 단백질 또는 유기 형광의 선택은 응용 프로그램 및 모델 시스템에 따라 달라집니다. 우리는 EGFP, YFP, 황수정, mEosFP, mEos2, tdEos, mOrange, PA-mCherry, 그리고 Dendra 12 등의 형광 단백질의 다양한, 테스트했습니다. 각 형광의 형광의 보존은 형광 단백질은 설명 방법을 사용하여 보존 할 수 있다는 제안, 비슷했다. 이 표현 때문에 우리는 C.에 잘 tdEos를 선택 elegans, 단백질은 tdEos에 융합 할 때 기능 유지, 그리고 또 다른 이유는 그사진 인증 특성은 PALM 현미경을위한 최적했다. 그러나, tdEos의 집합 또는 실패 표현식은 경우에 따라 12 관찰되었습니다. 응용 프로그램에 따라 다른 형광 더 적합한 수 있습니다. 대부분의 경우, 그것은 사진 활성 형광 단백질을 사용할 필요가 없습니다. 간단한 상호 형광 전자 현미경은 사진 활성 형광 단백질을 필요로하지 않습니다. GFP 또는 유기 염료는 회절 한도를 초과 섹션에 태그를 단백질에서 이미지 형광에 사용할 수 있습니다. 예를 들어, 하나는 이미지를 형광 현미경을 사용하여 neuropil의 축삭 및 이미징에 의한 전자 현미경에서 특정 축삭 형광 현미경에 형광과 형광 신호를 상관 관계 할 수 있습니다. 이러한 자극 방출 고갈 현미경 (전혀 두려움을 모르는 강인한) 12, 개별 분자 수익 (GSDIM) 13 다음 바닥 상태의 고갈 현미경과 같은 다른 슈퍼 해상도 기술,구조적 조명 현미경 (SIM) 14 사진 활성 형광 단백질이 필요하지 않습니다. 또한, 유기 염료 9,15,16 또는 형광 프로브 17 본질적인 속성을 사용 슈퍼 해상도 이미징 기술은 쉽게 적용 할 수 있습니다. 각 분자의 형광이 spatially과 시간적으로 분리되어 있기 때문에 PALM에서 분자의 수는 정량화 할 수 있습니다. 산화, undercounting, overcounting 및 overexpression : 그러나, 정량화 네 이유로 오해의 소지가있을 수 있습니다. 첫째, 형광 단백질의 일부가 변성 또는 샘플 처리 5,12 동안 산화 할 수 있습니다. 형광 신호의 ~ 90 %가 우리의 프로토콜에 고정하고 삽입을 통해 보존되었지만 표본이 sectioned되었으며 표면이 산소에 노출 후, 형광 단백질의 산화가 발생할 수 있습니다. 둘째, 사진 activatable 단백질의 활성화 확률이며, 따라서 여러 분자가 될 수 있습니다주어진 회절 제한된 장소 8 활성화. 여러 분자의 형광은 한 점으로 표시됩니다, 따라서 단백질의 총 수는 undercounted됩니다. 셋째, 비슷한 문제가 overcounting 될 수 있습니다. PALM에서 표백하여 각 형광 단백질은 지역화되어 있으며 다음 &quot;삭제&quot;. 그러나, 형광 단백질은 18 영구적으로 표백 않고 어두운 상태에서 돌아갈 수 있습니다. 이러한 분자 그런 다음 여러 번 계산 될 것입니다. 넷째, 태그 단백질은 transgenes으로 표현하고 자주 overexpression 될 수 있습니다 여러 복사본에 제시되어 있습니다. 따라서, PALM의 정량화는 지정된 위치에 분자의 수를 추정하지만, 정확하게 확인할 수 없습니다하는 데 사용할 수 있습니다. 전자 현미경과 PALM 이미지의 정렬은 또한 때문에 전자빔에 의한 빛과 전자 현미경과 왜곡의 해상도 차이에 도전 할 수 있습니다. 골드 입자는 단단히 LOC 제공전자 micrographs의 fiducial 마커를 alized. 그러나, 금 입자의 fluoresecence이 사진 활성화되어 있지 않습니다, 대형 회절 - 제한된 곳으로 나타납니다. 따라서, 전자 현미경을 통해 형광 이미지의 게재 위치는 추정치입니다. 왜곡은 또한 플라스틱 섹션 전자의 상호 작용에서 발생할 수 있습니다. 같은 GMA와 같은 아크릴 수지는 전자빔에 따라 덜 안정적이며, 플라스틱의 크기가 변경 될 수 있습니다. 이러한 상황에서 ultrastructure으로 형광을 정렬하면 fiducial 마커의 비 선형 변환이 필요할 수 있습니다.

<table border="1"><tbody><tr><td> 시약 또는 장비의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 고압 냉동기 </td><td> 상자를 </td><td> HPM 010 </td><td> EMPact과 Wohlwend에서 Leica 또는 hpf-01에서 HPM (100)는도 사용할 수 있습니다. </td></tr><tr><td> 자동 동결 대체 단위 </td><td> Leica </td><td> AFS 2 </td><td> AFS 1도 사용할 수 있습니다. </td></tr><tr><td> Zeiss PALM </td><td> Zeiss </td><td> ELYRA P.1 </td><td> 니콘과 Vutara 또한 상업적 PALM의 현미경을 판매하고 있습니다. </td></tr><tr><td> 전자 현미경 </td><td> 페이 </td><td> 노바 나노 </td><td> 기타 고해상도 SEM의 현미경을 사용할 수 있습니다. </td></tr><tr><td> 아세톤 </td><td> EMS </td><td> RT10016 </td><td></td></tr><tr><td> 에탄올 </td><td> 시그마 - 알드리치 </td><td> 459844-1L </td><td></td></tr><tr><td> 오스뮴 tetroxide </td><td> EMS </td><td> RT19134 </td><td></td></tr><tr><td> 칼륨 과망간산 염 </td><td> EMS </td><td> RT20200 </td><td></td></tr><tr><td> 소 혈청 알부민의 </td><td> 시그마 - 알드리치 </td><td> A3059-50G </td><td></td></tr><tr><td> Uranyl 아세테이트 </td><td> Polysciences </td><td> 21447-25 </td><td> 이 회사의 uranyl 아세트산의 pH는 약간 높다. </td></tr><tr><td> 글리콜 메타 크릴 레이트 (GMA) </td><td> SPI </td><td> 02630-AA </td><td> 낮은 산, TEM 등급. </td></tr><tr><td> N, N-디메틸-P-toluidine </td><td> 시그마 - 알드리치 </td><td> D9912 </td><td></td></tr><tr><td> Cryo 튜브 </td><td> Nalgene </td><td> 5000-0020 </td><td></td></tr><tr><td> 유리 병 </td><td> EMS </td><td> 72,632 </td><td></td></tr><tr><td> Aclar 필름 </td><td> EMS </td><td> 50425-10 </td><td></td></tr><tr><td> BEEM 캡슐 </td><td> EBSciences </td><td> TC </td><td> 폴리 프로필렌 </td></tr><tr><td> 3 / 8 &quot;디스크 펀치 </td><td> 테드 펠라 </td><td> 54,741 </td><td></td></tr><tr><td> 골드 나노 입자 </td><td> 마이크로 - nanospheres.com </td><td> 790122-010 </td><td> 2 배 농축 솔루션을 요청 </td></tr></tbody></table>

nano-fem: protein localization using photo-activated localization microscopy and electron microscopy

단백질의 분포를 매핑하면 셀에 단백질의 기능을 이해하는 것이 필수적입니다. 형광 현미경은 광범위하게 단백질 현지화에 사용하지만, subcellular 문맥 형광 이미지에 자주 결석입니다. Immuno-전자 현미경은 반면에, 단백질을 지역화 할 수 있지만, 대부분의 항원이 표본 표면에 노출되지 않기 때문에 기술은 호환 항체, 형태와 가난한 보존의 부족에 의해 제한됩니다. 상호 접근 방식 중 하나 immuno-형광이나 유전자 태그 단백질, 먼저 전체 셀에서 형광 이미지를 취득 할 수 있습니다. 예제는 다음 고정 및 전자 현미경에 내장하고, 이미지가 1-3 상관이됩니다. 그러나, 낮은 해상도 형광 이미지와 fiducial 마커의 부족은 단백질의 정확한 현지화를 배제. 또한, 형광 이미징은 플라스틱의 표본을 보존 한 후에 수행 할 수 있습니다. 에이 방법은 블록 sectioned이며, 형광 이미지와 동일한 섹션의 전자 micrographs는 4-7를 서로 관련되어 있습니다. 그러나, 상관 이미지에 빛의 회절 한계는 개별 분자의 위치를​​ 가린다하고, 형광은 종종 세포의 경계 넘어 확장합니다. 나노 해상도 형광 전자 현미경 (나노 FEM)이 사진 활성 현지화 현미경 (PALM)과 전자 현미경을 사용하여 이미징 같은 섹션으로 나노 스케일의 단백질을 현지화하기 위해 설계되었습니다. PALM는 영상 개인 형광 단백질에 의해 회절 한계를 극복하고 이후 각 형광 현장 8-10의 중심을 매핑. 우리는 다섯 단계에있는 나노 FEM 기술을 설명합니다. 먼저, 예제는 고정 및 태그 단백질의 형광을 보존 조건을 사용하여 포함하고 있습니다. 둘째, 수지 블록 장착 ultrathin 세그먼트 (70-80 nm 정도)에 sectioned 아르커버 유리에. 셋째, 형광은 Zeiss의 PALM 현미경을 사용하여 이러한 섹션에 이미징된다. 넷째, 전자 고밀도 구조는 스캔 전자 현미경을 사용하여 이러한 동일한 섹션에 이미징 있습니다. 다섯째, 형광 및 전자 micrographs는 fiducial 마커 등의 금 입자를 사용하여 정렬된다. 요약하면, 휘황 태그 단백질의 subcellular 지방화는 약 1 주일 나노 미터 해상도로 결정될 수있다.

Watch this Scientific Journal Video about Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy at JoVE.com

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

우리는 전자 micrographs에 휘황 태그 단백질을 지역화하는 방법을 설명합니다. 형광 먼저 ultrathin 섹션에 사진 활성 현지화 현미경을 사용하여 지역화되어 있습니다. 이러한 이미지는 다음 같은 섹션의 전자 micrographs으로 정렬되어 있습니다.

나노 FEM : 사진 활성화 된 현지화 현미경과 전자 현미경을 사용하여 단백질 현지화

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

nano-fem-protein-localization-using-photo-activated-localization

Research

JoVE Journal

Biology

15.9K 조회수.  University of Utah. 우리는 전자 micrographs에 휘황 태그 단백질을 지역화하는 방법을 설명합니다. 형광 먼저 ultrathin 섹션에 사진 활성 현지화 현미경을 사용하여 지역화되어 있습니다. 이러한 이미지는 다음 같은 섹션의 전자 micrographs으로 정렬되어 있습니다.

비디오: Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

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생물학

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

멀티 플렉스 면역 조직 화학적 염색 및 멀티 스펙트럼 영상을 사용하여 단백질 발현 및 공동 현지화의 정량

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and &#62; 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

입체 슈퍼의 간섭 광 활성화 현지화 현미경으로 F-액틴 필라멘트의 해상도 현미경 (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

살아있는 포유류 세포의 세포에서 단일 막 단백질의 다 색 지역화 현미경

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

고압 냉동, 전자 레인지를 이용한 콘트라스트 향상, 그리고 볼륨 이미지에 대 한 포함 하는 최소한의 수 지에 의해 생물학 견본 준비

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

전 생체 내 공초점 현미경을 사용하여 림프절 구조 및 세포 국소화 시각화

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

초구조적으로 보존된 세포 내 막 단백질 국소화의 전자 현미경 분석을 위한 CryoAPEX 방법

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

단일 분자 국소화 현미경 검사를 사용하여 인간 iPSC 파생 심근세포에서 α-액티닌 네트워크 분석

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

새로운 로봇 시스템, 스포티폰을 사용하여 시간 해결 연구를 위한 극저온 전자 현미경 그리드 준비

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.