Name: 3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles
Uploaded: 2014-10-01T13:00:00.000+00:00
Duration: 11 min 28 s
Description: Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

1 샘플 준비 <ol><li> 10 % 소 태아 혈청 및 페니실린 스트렙토 마이신 50 ㎍ / ml의 100 IU / ㎖로 보충 된 DMEM을 사용하여 조직 배양 플라스크에 CHOK1 세포를 유지한다. 37 ° C에서 5 % CO 2 가습 인큐베이터에있는 세포를 품어. </li><li> 수확 후 0.16 mm의면 두께 14mm 직경 마이크로 웰에 CHOK1 세포를 플레이트. 60~70% 자랄 때 주변의 이미징을위한 최적의 밀도에 대한 종자 세포. </li><li> 37 ° C에서 하룻밤 세포를 품어, 5 % CO 2. 녹색과 튜 불린 추적기 녹색의 150 nm의 Lysotracker DND26의 50 nm의를 포함하는 용액에 세포를 HBSS (행크 버퍼 식염수)의 셀을 세 번 세척하고, 부화. 37 ° C에서 1 시간 동안 세포를 품어. 선택 단계 : 부화하기 전에, 미토콘드리아 매트릭스 염색 염료 (25 nM의)와 추가로 얼룩을 수행합니다. </li><li> 어떤 언 바운드 염료를 제거 HBSS에있는 세포를 세 번 씻으십시오. IMAG 이전에 신선한 DMEM 성장 매체를 추가세포를 보내고. </li></ol> 2 현미경 구성 비디오 프로토콜에 기재된 입자 추적 현미경 시판 도립 현미경 (도 4)의 프레임에 조립된다. 3D 궤도 입자 추적을위한 그러나 상용 모듈을 사용할 수 있습니다. <ol><li> 690 nm의 1,040 nm의 사이에 조정 가능한 출력 파장 범위, 사 펨토초 레이저의 여기 광원 : 코 히어 런트 카멜레온 - 울트라 II 티를 사용합니다. </li><li> 레이저 빔이 IR 코팅 거울을 사용하여 현미경의 후면 포트에 정렬되어 있는지 확인합니다. 레이저 빔은 방해석 선형 편광자 다음 회전 반 파장 판을 이용하여 감쇠된다. 샘플에서의 평균 전력은 0.5과 2 mW의 사이가되도록, 빔을 감쇠. 주 : 레이저 빔이 시료면에 집속 빔의 위치 및 궤적의 제어를 허용 검류계 미러 구동 모터의 쌍에 의해 반사된다. 에서짧은 패스 다이크로 익 미러에 반사 한 후 현미경 대물의 후방에 들어가기 전에, 빔 익스팬더 통과 갈바 노 미러의 출사 평행 레이저 빔 확장 전형적인 구성 (10X). </li><li> 광로에 높은 개구 물 대물 (60X, NA 1.2)를 배치하여 샘플에서 형광 광을 수집한다. 하나 또는 두 개의 채널 구성에 원하는 발광 파장에 의한 형광 필터 큐브를 선택. 상기 방출 된 형광 스펙트럼의 범위를 선택하는 발광 대역 통과 필터를 사용한다. </li><li> 전동 무대에 샘플을 놓고 객관적 압전 컨트롤러를 사용하여 현미경 목표의 미세한 움직임을 조정합니다. </li><li> 신호 판별 및 디지털 I / O 데이터 수집 카드로 전송되는 광전자 증 배관 튜브에 필터 정육면체로부터의 광을 직접. NOTE : 컴퓨터 파형은 I / O 카드의 아날로그 출력 및 제공하기 아르 생성스캐너에 에드는 전자를 제어 할 수 있습니다. 광전자 증 배관 및 스캐너로 출력 신호에서 광자의 카운트 입력은 측정 된 형광 SimFCS 역학 소프트웨어를위한 실험실에 의해 I / O 카드를 통해 제어된다. </li></ol> 3 이미징 <ol><li> 무대에서 세포를 놓고 투과광 조명을 사용하여 초점을 맞 춥니 다. </li><li> 염료를 혼입 한 세포를 식별하고 기본 미세 소관을 시각화하는 래스터 스캔 영상으로 전환. 소포 운동이 존재하는 초기 영역을 식별합니다. </li><li> 고립 된 소포가 확인되면, 궤도 추적에 대해 다음 매개 변수를 설정합니다 : <ol><li> 입자의 크기에 따른 추적되는 원형 주사의 크기를 정의하기 위해 궤도 반경을 선택한다. 포인트 이미 터, 감도 및 응답을 최적화 여기 빔의 점 확산 함수 (PSF)의 허리 동일한 궤도의 반경을 설정한다. </li><li> 축 설정축 방향을 따라 파티클 위치 국산화 수행이 궤도 사이의 거리를 정의하는 거리. 1.5-3 배 PSF 허리에 축 방향의 거리를 설정합니다. </li><li> 또한 광 표백 속도를 결정할 것이다 궤도의 각 시점에 소비되는 시간을 설정하는 입자의 밝기에 따라 체류 시간을 정의한다. 10 ~ 100 마이크로 초 사이의 체류 시간을 사용합니다. </li><li> 4-32 밀리의 순서 궤도주기를 산출하고, 입자의 위치를​​ 결정하기위한 높은 시간 해상도를 제공하기 위해 64 또는 128로 각각의 궤도에있는 포인트의 수를 설정한다. </li></ol></li><li> DC보기 래스터 스캔 이미지에서 커서 선택을 통해 그래픽 사용자 인터페이스를 통해 입자에 빔 중앙 위해 거울에 전달 상쇄 신호를 변경한다. </li><li> 궤도 검사에 래스터 이미지에서 현미경 모드를 전환하여 추적을 시작합니다. </li><li> 피드백 및 데이터 수집을 활성화합니다. </li></ol> 4 Traject모리 분석 <ol><li> 궤도와 함께 &#39;강도 카펫&#39;의 형태로 각 시점의 각 시점에서 수집 된 형광을 표시하도록 소프트웨어를 사용한다. 카펫을 따라 수집 된 강도는 다른 밝은 물체로 입자의 상호 작용에 대한 정보를 제공합니다. 물론 시계열의 형태로 시간에 걸쳐 광전자 증 배관으로부터 수집 형광 강도 (x, y 스캐너 및 Z의 -piezo의 시간 동안 DC 변위 즉) 궤적 정보를 모두 표시하도록 소프트웨어를 사용한다. </li><li> 궤도에 대한 관심 만 지역을 선택하는 시계열 표현하고 &#39;강도 카펫의 정보를 사용합니다. </li><li> 입자 궤적의 선택된 부분의 2D 투영을 표시 할 소프트웨어를 사용합니다. 입자의 형광 강도에 따라 색상 코드로 궤도를 적절한 컨트롤을 선택합니다. </li><li> 페이지를 표시하는 옵션을 선택합니다형광 강도에 따라 3D로 문서 궤도, 색상 코드 그것. 미세 소관을 따라 리소좀 운동의 특징을 시각화하기 위해 컨트롤을 사용하여 3D의 궤도를 돌립니다. </li></ol>

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The authors have nothing to disclose.

높은 시간 분해능으로 3 차원 형광 입자의 궤적을 달성 지난 년 동안 형광 현미경 기술과 계장의 엄청난 진행됨에도​​ 불구 분야에서 도전을 유지하고있다. 높은 시간 해상도가 이차원 추적 입자를 달성 한 경우, 축 방향에 확장은 전형적으로 시스템 (10)의 주파수 응답에서의 급격한 감소를 가져온다. 이 동영상 프로토콜에서는 이광자 현미경을 사용하여 높은 시?...

방법 중 다수의 현미경을 사용 입체적으로 형광 발색 입자를 추적하는 날짜로 개발되어왔다. 대부분의 접근은 일반적으로 이상적인 축 방향으로 트래킹을 달성하기 위해 현미경 출사 광학계의 정의 수정과 조합이 차원에서 추적하기 적합한 고속 카메라의 사용에 의존한다. 이 프로세스가 적절한 시간 해상도 (10Hz의)는 일반적으로 시간 소모적이며, 얻을 수 있지만, 종래의 입자가있다 추적되는 경우에만, Z-스택의 시계열을 수행하여 형광체 입자를 추적 할 수있다 (촛점 또는 두 개의 광자 중) 레이저 주사 현미경 액티브 피드백 메커니즘 (2)에 의해 작은 래스터 촬상 영역의 중심에 있었다. 록킹에서 입자로의 개념은 궤도 추적 방법의 기초이다. 대신 래스터의 원형 궤도가 형광 입자의 주위에 수행되는 검사합니다. 따라서 형광의 강도궤도 정확하게 입자의 총수 4 편재. 입자의 국부적 위치는 현미경 스캐너 재 중심 위치에 입자 궤도를 작동하는 데 사용될 수있다. 현미경의 갈바 노 스캐너는 아날로그 전압에 의해 구동된다. 이 전압의 AC 성분은 사인 및 원 궤도를 수행 할 수 있도록 X 및 Y 스캐닝 미러에 적용 코사인 파 즉, 집속 레이저 빔의 궤도를 수행 할 수 있습니다. 신호의 DC 오프셋은 궤도의 중심 위치를 변경하는 것을 용이. 궤도주기가 결정되며, AC 신호 파형이 구성되면, 피드백 시스템은 신호의 단지 DC 성분을 업데이트 할 필요가있다. 검출기로부터 수집 된 형광 신호를 판독 할 수 소프트웨어는 DC 전원을, 스캐너 및 검출기를 제어하기 위해 입자의 위치를​​ 계산하고 갱신 할 필요설정합니다. 궤도 파라미터 (크기 및 타이밍)의 신중한 선택을 묘화 성공적인 피드백에 필요한 이들 파라미터는 추적 할 필요가 형광 물질의 특성에 기초하여 조절 될 필요가있다. 기술의 물리적 및 수학적 기초는 2D 및 3D 애플리케이션 모두에 대해 과거 4,5에 기재 하였다. 이 프로토콜에서는 간단히 셋업의 주요 하드웨어 구성 요소를 설명하고, 더욱 구체적으로 파라미터의 선택과 시료 전처리는 살아있는 세포 내의 높은 시간적 해상도 리소좀의 변위 다음 우릴 있도록 일반적인 실험에 필요한.

<table><tbody><tr><td>Lysotracker DND 26</td><td>Life Technologies</td><td>L-7526</td><td></td></tr><tr><td>Tubuline tracker Green</td><td>Life Technologies</td><td>T34075</td><td></td></tr><tr><td>Mitotracker RED</td><td>Life Technologies</td><td>M7512</td><td></td></tr><tr><td>Coherent Chamelon- ultra II TI</td><td>Coherent</td><td></td><td></td></tr><tr><td>Glan Taylor Calcite Polarizer</td><td>Melles Griot</td><td>03PTA001</td><td></td></tr><tr><td>Galvanometer-motor mirror</td><td>Cambridge Technologies</td><td>M 6350</td><td></td></tr><tr><td>Dichroic mirror</td><td>Chroma Technologies</td><td>700 DCSPXR</td><td></td></tr><tr><td>Motorized stage</td><td>ASI</td><td>MS2000</td><td></td></tr><tr><td>Piezo&amp;nbsp;</td><td>PI</td><td>P721-LLQ</td><td></td></tr><tr><td>Photomultiplier tube</td><td>Hamamatsu</td><td>H7422P-40</td><td></td></tr><tr><td>Data acquisition card</td><td>IO tech</td><td>PCI 1128-4000</td><td></td></tr><tr><td>Imaging software</td><td>LFD</td><td>Global for images-SimFCS</td><td></td></tr></tbody></table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

이 동영상 프로토콜의 목적은 변형 된 이광자 현미경을 사용하여 1 입체 형광 입자 궤도 추적 실험을 수행하고 분석하는 방법을 설명하는 것이다. 종래의 접근 방식 (래스터 스캔 또는 프레임들의 스택에 기초하여 넓은 시야)에 대향 된 바와 같이, 3D 궤도 추적 지역화 및 높은 공간 (10 nm의 정확도) 및 시간 해상도 (50 Hz에서의 주파수 응답)의 3 차원 변위에 따라 허용 마이크론이 수백의 길이 비늘에 이동 형광 입자. 이 방법은 추적 된 대상물 주위에 원형 궤도를 수행하기 위해 두 개의 광자 레이저 스캐닝 현미경의 하드웨어를 제어하는​​ 피드백 알고리즘을 기반으로한다 : 피드백기구의 변위를 제어하여 중심에 형광 물체를 유지 주사 빔 3-5. 이 기술의 장점을 설명하기 위해, 우리는 알 내에서 빠르게 움직이는 세포 기관, 리소좀을 따라iving 셀 6,7. 세포는 표준 프로토콜에 따른 도금 및 상업적 리소좀 염료를 사용하여 염색 하였다. 우리는 살아있는 세포의 내부 3D 궤도 추적 실험을 수행하기 위해, 간단히 하드웨어 구성과보다 상세히 제어 소프트웨어를 논의한다. 우리는 상세히 주사 현미경을 제어하고 입자 주위 닫힌 궤도 빔의 움직임을 사용하기 위해 필요한 파라미터들을 논의한다. 우리는이 방법이 효과적으로 생균 내에 3 차원 미세 소관을 따라 표시된 리소좀의 빠른 움직임을 추적 할 수있는 방법을 보여줌으로써 결론. 리소좀은 일반적으로 미세 소관 네트워크 8 따라 모션을 지시 표시, 0.4-0.5 μM / 초 범위의 속도로 이동할 수있다.

이 프로토콜에 따라 고속 3D 단일 입자 추적은 형광 표지 리소좀의 변위를 추적하는 개질 이광자 현미경을 이용하여 살아있는 세포 내부에서 수행 될 수있다. 실험 수행 엔도 좀의 성숙 과정 9 후 셀 내부 이동 절연 리소좀 추적 이루어져있다. 리소좀은 녹색 형광 염료를 사용하여 염색하고 2 광자 여기 악용 930 nm에서 여기시켰다. 우리의 데이타는 32 밀리 초 (도 1A)의 시간 해상...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

수정 된 두 개의 광자 현미경에서 3D 궤도 추적 : 세포 내 소포의 추적에 응용

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Research

JoVE Journal

Bioengineering

10.2K 조회수.  University of California, Irvine. In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. 

비디오: 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

네 차원 형광 이미징과 신경 말단 생활에 엔도 좀 역학의 시각화

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

연구

신경과학

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

초파리 배아에서 지질 함유 소기관의 세포 골격 의존성 밀매 시각화

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

발생학

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Spectrally 해결된 두 광자 현미경을 사용하여 G 단백질 결합 수용체 상호 작용의 생체내 부량에서

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

생물학

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

실시간 3D 단일 입자 추적에 대 한 프로토콜

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

생체공학

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

형광 프로브를 이용한 배양 된 뉴런의 엔도 좀과 리소좀 기능의 정량화

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

Vivo에서 단일 분자 초파리 연 접 모터 신경 맨끝에 추적

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

효모의 4D 현미경 검사법

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

3 차원에서 세포 외 소포의 직접 스토카스틱 광학 재건 현미경 검사

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Source: Tsuruta, F., et al. <a href="https://app.jove.com/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging(형광 이미징을 사용한 뉴런의 소포 운동성 시각화)

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...