YW 및 PK는 감사 PK (NRF-NRFF-2011-04 및 NRF2012NRF-CRP001-084)에게 수여 싱가포르 국립 연구 재단에서 자금 지원을 인정합니다. 우리는 또한 인프라 지원을위한 MBI 개방 실험실과 현미경의 핵심 시설을 주셔서 감사합니다.

<ol>
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저자는 공개 아무것도 없어.

도 1a에 도시 된 바와 같이 iPALM의 광학계는, 4 π 이중 대향 대물 설계에 기초한다. 표 1에 이전 (23) 설명 및 나열된 설정은 사용자 정의 가공 및 상업 모두 광학 기계 부품을 사용하여 구성된다. 우리의 설치뿐만 아니라, 하워드 휴즈 의학 연구소 (HHMI)는 Janelia 연구 캠퍼스에서 고급 이미지 센터에서 과학계에 액세스 할 수있는 시스템을 호스팅합니다. ?...

복잡한 세포 구조의 시각화는 오랫동안 생물 통찰력과 발견을 통합하고있다. 형광 현미경은, 그 해상력이 ~ 200 화상면에 나노 (X, Y, 또는 측 방향 치수) 및&gt; 500 nm의 광학 축 방향 (Z, 또는 축 방향 치수)에 회절 고분자 특이성 화상 세포 제한 할 수 있지만 1, 2. 따라서, 미세 기능 관찰 역사적 전자 현미경 (EM)로 제한되어왔다. 100 nm의 범위 1-6 - 다행히, 슈퍼 해상도 현미경의 최근 발전은 10에서 공간 해상도를 가능하게,이 제한을 회피했다. 특히, 슈퍼 해상도는 같은 PALM (광 활성화 현지화 현미경) 4, FPALM은 (형광 광 활성화 현지화 현미경) 5 (d)에 STORM (직접 확률 광학 재건 현미경) 6, 7, PAINT (같은 약어로 알려진 단일 분자 현지화에 기반 접근 방법 포이미징 나노 지형) 8 INT의 축적은, GSDIM (접지 상태 고갈 현미경 9) 개별 분자 반환 한 다음, 또는 SMACM (단일 분자 액티브 제어 현미경) (10),뿐만 아니라 그들의 3 차원 (3D) 구현, 간섭 PALM (iPALM) 11 3D-STORM (12)는 신경 세포의 축삭을, 다수의 생물학적 구조의 나노 조직에 새로운 통찰력을 드러내는 포함 귀중한되었습니다 및 초점 유착 14, 15, 세포 - 세포 접합 (16), 원자력이 17 모공, 13 시냅스 및 중심체 18 ~ 20 등이 있습니다. 슈퍼 해상도 현미경은 잠재적으로 유용하는 세포의 또 다른 미세 기능은 액틴 세포 골격이다. 셀 피질 필라멘트 (F) 액틴의 복잡한 그물 세공은 세포 형태 및 기계적 특성 (21)의 제어에 중요한 역할을한다. 조직 오F의 F-액틴 적극적 동적 강하게 중합, 가교, 회전율, 안정성 및 네트워크 토폴로지 (22)에 영향을 미치는 수많은 조절 단백질 비록 조절된다. 그러나, F - 굴지 그물 세공 구조의 특성은 세포 과정의 다양한 범위, 기존의 회절 제한 광학 현미경으로 자신의 관찰을 방해은 F-액틴 필라멘트의 작은 크기 (~ 8 ㎚)에 기계적인 통찰력에 대한 중요하지만, 따라서 액틴 미세 구조의 시각화 종래 독점적 EM에 의해 수행되었다. 여기서는, 접착 포유류 세포에서 액틴 세포 골격 F-시각화 3D 11,23에 매우 높은 정밀도 기능을 활용할 iPALM 초 해상 현미경 기술을 사용하는 프로토콜을 기술한다. iPALM 악기는 매우 전문 있지만, 이러한 장비를 설정하는 방법에 대한 지시는 최근 23 설명되어있는 호 주최 iPALM 현미경에 액세스하는 동안구 휴즈 의학 연구소는 또한 최소의 비용으로 연구 커뮤니티에 제공하고있다. 또한, 본원에 기재된 시료 제조 방법은 더 광범위하게 사용할 수있는 등의 점 확산 함수 (PSF)의 난시 디 포커싱 기반으로하는 다른 3D 슈퍼 해상도 방법, (12) 또는 이중면 검출부 (24)에 직접적으로 적용될 수있다. 우리는 일반적으로 단일 분자 현지화 기반의 초 고해상도 현미경을위한 필요한 성분이 충족되어야 단일 분자 현지화 기반의 초 고해상도 현미경을위한 세 가지 핵심 요구 사항을 수있는 광전 환성 형광 25이므로주의 : ⅰ) 높은 단일 분자 배경 신호의 밝기 및 대비 상대; ⅱ) 소정의 이미지 프레임 내의 단일 분자의 희박한 분포; 또한 나이 퀴 스트 - 샤로 알려진 기본 구조 (의 프로필을 캡처하기에 충분한 라벨 및 ⅲ) 높은 공간 밀도nnon 샘플링 기준) 26. 따라서, 만족스러운 결과를 위해, 형광체의 강조 photoswitching을 최적화하고 기본 미세 구조를 보존 할뿐만 아니라, 실험 기기 및 수집 측면하는 시험편의 적절한 준비를 모두 동등하게 배치되어야한다.

<table><tbody><tr><td>optical table</td><td>Newport, CA</td><td>RS4000</td><td>iPALM, installed on 4 Newport Stabilizer vibration isolators</td></tr><tr><td>vibration isolator for optical table</td><td>Newport, CA</td><td>S-2000</td><td></td></tr><tr><td>laser-642</td><td>Newport, CA</td><td>1185055</td><td>output power=100 mw</td></tr><tr><td>laser-561</td><td>Newport, CA</td><td>1168931</td><td>output power=200 mw</td></tr><tr><td>laser-488</td><td>Newport, CA</td><td>1137970</td><td>output power=200 mw</td></tr><tr><td>laser-405</td><td>Newport, CA</td><td>1142279</td><td>output power=100 mw</td></tr><tr><td>broadband dielectric mirrors</td><td>Thorlabs, NJ</td><td>BB1-E02</td><td>laser combiner</td></tr><tr><td>dichroic beamsplitter</td><td>Semrock, NY</td><td>LM01-427-25</td><td></td></tr><tr><td>acousto-optic tunable filter</td><td>AA Opto-Electronic, France</td><td>AOTFnC-VIS-TN</td><td></td></tr><tr><td>Linear polarizer</td><td>Newport, CA</td><td>05LP-VIS-B</td><td></td></tr><tr><td>baseplate</td><td>local workshop</td><td>customized</td><td></td></tr><tr><td>turning mirror (22.5&amp;deg;)</td><td>Reynard Corpporation, CA</td><td>customized</td><td>22.5&amp;deg; mirror</td></tr><tr><td>motorized optic mounts</td><td>New Focus, CA</td><td>8816</td><td></td></tr><tr><td>motorized XYZ translation stage</td><td>Thorlabs, NJ</td><td>MT3/M-Z6</td><td>sample holder</td></tr><tr><td>T-Cube DC servo motor controller</td><td>Thorlabs, NJ</td><td>TDC001</td><td></td></tr><tr><td>Piezo Phase Shifter</td><td>Physik Instrumente, Germany</td><td>S-303.CD</td><td></td></tr><tr><td>objective lens</td><td>Nikon, Japan</td><td>MRD01691</td><td>objective. Apo TIRF 60X/1.49 oil</td></tr><tr><td>translation stage</td><td>New Focus, CA</td><td>9062-COM-M</td><td></td></tr><tr><td>Pico Motor Actuator</td><td>New Focus, CA</td><td>8301</td><td></td></tr><tr><td>rotary Solenoid/Shutter</td><td>DACO Instruments, CT</td><td>5423-458</td><td></td></tr><tr><td>3-way beam splitter</td><td>Rocky Mountain Instruments, CO</td><td>customized</td><td>beamsplitter</td></tr><tr><td>Piezo Z/Tip/Tilt scanner</td><td>Physik Instrumente, Germany</td><td>S-316.10</td><td></td></tr><tr><td>motorized five-axis tilt aligner</td><td>New Focus, CA</td><td>8081</td><td></td></tr><tr><td>Picmotor ethernet controller</td><td>New Focus, CA</td><td>8752</td><td></td></tr><tr><td>Piezo controllers/amplifier/digital operation module</td><td>Physik Instrumente, Germany</td><td>E-509/E-503/E-517</td><td></td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-523/20</td><td>filters</td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-588/21</td><td></td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-607/30</td><td></td></tr><tr><td>band-pass filter</td><td>Semrock, NY</td><td>FF01-676/37</td><td></td></tr><tr><td>notch filter</td><td>Semrock, NY</td><td>NF01-405/488/561/635</td><td></td></tr><tr><td>motorized filter wheel with controllter</td><td>Thorlabs, NJ</td><td>FW103H</td><td></td></tr><tr><td>EMCCD</td><td>Andor, UK</td><td>DU-897U-CSO-#BV</td><td>3 sets</td></tr><tr><td>Desktop computers for controlling cameras and synchronization</td><td>Dell</td><td>Precision T3500</td><td>PC, 4 sets</td></tr><tr><td>coverslips with fiducial</td><td>Hestzig, VA</td><td>600-100AuF</td><td>sample preparation. fiducial marks with various density and spectra available</td></tr><tr><td>fibronectin</td><td>Millipore, MT</td><td>FC010</td><td></td></tr><tr><td>paraformaldehyde</td><td>Electron Microscopy Sciences, PA</td><td>15710</td><td>fixation. 16%</td></tr><tr><td>glutaraldehyde</td><td>Electron Microscopy Sciences, PA</td><td>16220</td><td>25%</td></tr><tr><td>triton X-100</td><td>Sigma aldrich, MO</td><td>T8787</td><td></td></tr><tr><td>HUVEC cells</td><td>Life Technologies, CA</td><td>C-015-10C</td><td></td></tr><tr><td>Medium 200</td><td>Life Technologies, CA</td><td>M-200-500</td><td></td></tr><tr><td>Large Vessel Endothelial Factors</td><td>Life Technologies, CA</td><td>A14608-01</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td></td><td>14190367</td><td></td></tr><tr><td>Pennicillin/Streptomycin</td><td></td><td>15140122</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies, CA</td><td>25200056</td><td></td></tr><tr><td>PIPES</td><td>Sigma aldrich, MO</td><td>P1851</td><td>PHEM</td></tr><tr><td>HEPES</td><td>1st base, Malaysia</td><td>BIO-1825</td><td></td></tr><tr><td>EGTA</td><td>Sigma aldrich, MO</td><td>E3889</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Millipore, MT</td><td>5985</td><td></td></tr><tr><td>Alexa Fluor 647 Phalloidin</td><td>Invitrogen, CA</td><td>A22287</td><td>staining</td></tr><tr><td>sodium borohydride (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma aldrich, MO</td><td>480886</td><td>quenching</td></tr><tr><td>glucose</td><td>1st base, Malaysia</td><td>BIO-1101</td><td>imaging buffer</td></tr><tr><td>glucose oxidase</td><td>Sigma aldrich, MO</td><td>G2133</td><td></td></tr><tr><td>catalase</td><td>Sigma aldrich, MO</td><td>C9322</td><td></td></tr><tr><td>cysteamine</td><td>Sigma aldrich, MO</td><td>30070</td><td></td></tr><tr><td>Epoxy</td><td>Thorlabs, NJ</td><td>G14250</td><td></td></tr><tr><td>vaseline</td><td>Sigma aldrich, MO</td><td>16415</td><td>sample sealing</td></tr><tr><td>lanolin</td><td>Sigma aldrich, MO</td><td>L7387</td><td></td></tr><tr><td>parafin wax</td><td>Sigma aldrich, MO</td><td>327204</td><td></td></tr><tr><td>Immersion oil</td><td>Electron Microscopy Sciences, PA</td><td>16915-04</td><td>imaging. Cargille Type HF</td></tr></tbody></table>

three-dimensional super resolution microscopy of f-actin filaments by interferometric photoactivated localization microscopy (ipalm)

형광 현미경은 세포 내 특정 생체 분자를 직접 시각화 할 수 있습니다. 그러나, 종래의 형광 현미경, 공간 해상도는 광축을 따라 결상면과&gt; 내지 500nm에서 ~ 200 nm의 회절에 의해 제한된다. 결과적으로, 형광 현미경은 오랫동안 심각한 세포 내 미세 기능의 관찰에 한정되었다. 초 해상 현미경 방법의 최근 발전은 이러한 한계를 극복하고있다. 특히, 광전 환성 형광체의 출현 분자 길이 스케일에 접근 해상력을 제공하는 기반 지역화 초 해상 현미경을 가능하게한다. 여기서는 간섭계 광 활성화 지역화 현미경 (iPALM)이라는 단일 분자 파악 현미경 다상 간섭에 기초하여 입체 초 해상 현미경 법의 적용을 설명한다. 이 방법은에 거의 등방성 해상도를 제공세 가지 차원에서 20 나노 미터의 순서. iPALM 악기의 시료 준비 및 운영을 포함하여 사상 액틴 세포 골격을 시각화 프로토콜, 여기에 설명되어 있습니다. 이 프로토콜은 세포에서 다른 미세 구조 기능 연구에 쉽게 적응과 교훈입니다.

iPALM 중요 요건은 광학 시스템의 정렬, 등록 및 교정된다. 추출 Z 좌표에 대한 이들 3 방향 빔 스플리터 내에서 필요한 적절한 간섭을 보장하기 위해 필요하다. 지속적인 모니터링을 사용하려면, 형광의 일정 지점 소스가 필요합니다. 이것은 형광 금 또는 그 포토 루미 국소 표면 플라즈몬 공명 (LSPR)에서 발생하는 이중 금속 나노 입자 (23)를 사용하여 달성 될 수?...

Watch this Scientific Journal Video about Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM at JoVE.com

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

우리는 접착 포유류 세포에서 액틴 세포 골격의 이미징에 간섭 광 활성화 지역화 현미경 (iPALM), 3 차원 단일 분자 지역화 초 해상 현미경 법의 적용 프로토콜을 제시한다. 이 방법은 다른 기존의 회절 제한 광학 현미경으로 해결되지 않은 남아있을 것입니다 나노 구조적 특징의 빛 기반 시각화 할 수 있습니다.

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 ...

three-dimensional-super-resolution-microscopy-f-actin-filaments

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

10.7K 조회수.  South University of Science and Technology of China, Shenzhen.  우리는 접착 포유류 세포에서 액틴 세포 골격의 이미징에 간섭 광 활성화 지역화 현미경 (iPALM), 3 차원 단일 분자 지역화 초 해상 현미경 법의 적용 프로토콜을 제시한다. 이 방법은 다른 기존의 회절 제한 광학 현미경으로 해결되지 않은 남아있을 것입니다 나노 구조적 특징의 빛 기반 시각화 할 수 있습니다. 

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

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

형광등을 통해 F - 굴지 역학 라이브 셀 이미징은 현미경을 스페클 (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

연구

생물학

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

구조화 조명 현미경 데이터의 시공간적 이미지 상관 분광학에 의해 정량화 T 세포에서 대뇌 피질 굴지 흐름

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

면역학 및 감염병학

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

동시 간섭 반사 및 전체 내부 반사 형광 현미경 검사 이미징 동적 미세 소관 및 관련 단백질

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

생화학

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

미세 유체 공학 및 형광 현미경을 사용하여 단일 액틴 필라멘트 및 번들의 조립 역학 연구

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

정확한 세포 표현과 정확한 단백질 국소화를 위한 세균 세포의 3차원 이미징

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

세균 본부 기계의 슈퍼 해상도 이미징

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

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 ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

고속 3D 구조 조명 현미경을 사용하여 라이브 박테리아의 Cytokinetic Z 반지의 슈퍼 해상도 이미징 (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells