처음 Sive 연구실에서이 기술을 개발 박사 제니퍼 구츠맨 많은 감사드립니다. 친절 플라스미드 인코딩 CAAX - GFP의 mRNA를 제공 데이나 파버 암 연구소 - 보스턴, MA에서도 JB 그린 감사합니다. 공촛점 이미지는 화이트 헤드의 WM 켁 재단 생물 이미징 시설에서 실시되었다. HS는 NIHMH70926에 의해 지원됩니다. EG는 NSF 미리 박사 친교에 의해 지원됩니다.

1. 사출에 대한 mRNA 준비 <ol><li> 이 절차에 사용되는 mRNA는 플라스미드 인코딩 CAAX - eGFP (memGFP) mRNA에서 베꼈는데 있습니다. 먼저 플라스미드는 구츠맨 외, 2008에 따라 linearize. </li><li> 다음 mMessage mMachine 키트를 사용 memGFP mRNA를 고쳐 쓰다. </li><li> -80에서 결과 1μg/μl으로 mRNA, 나누어지는 및 상점을 희석 ° C. </li><li> 한 세포 단계에서 배아 차선의 넓이와 1퍼센트 아가로 오스의 사출 금형을 준비합니다. </li><li> 주사 전날에, 여성에서 남성을 분리 교배 새장을 설정합니다. </li><li> 주사 당일, 주사를 준비하는 micropipette의 풀러를 사용하여 모세 바늘을 가져옵니다. </li></ol> 2. mRNA를 주입 <ol><li> 주사 당일, 얼음 1μg/μl의 memGFP의 mRNA의 나누어지는를 녹여. </li><li> 200ng/μl의 최종 농도로 물에 mRNA 1:5 희석, 얼음 계속. </li><li> 200ng/μl에서 준비 memGFP의 mRNA의 1μl와 모세관 바늘을로드합니다. </li><li> 장소는 가스 공급 microinjector에 연결된 micromanipulator에 바늘을로드. </li><li> 1nl로 사출 볼륨을 조정합니다. </li><li> 전날부터 짝짓기 새장에 설치된 야생 종류의 물고기에 디바이더를 당겨. </li><li> 즉시 그들이 낳은 그대로 배아를 수집합니다. 동양들을 멀리 micromanipulator의 측면에 하나의 세포로 배아 매체 (웨스 터, 1995)이 적용 아가로 오스의 사출 금형 인치 </li><li> chorion을 통해 삽입하여 mRNA가 하나의 세포에 직접 입금됩니다 그러한 노른자. 실험 당 약 50 배아를 주사. 세포가 나뉘어져있다면 삽입하지 마십시오. </li><li> 28 ° C 야간 배아 매체 품어 배아 </li></ol> 3. 마운트 및 이미징 태아 <ol><li> 배아를 마운트 및 이미지하려면, 관심있는 시점 전에 stereomicroscope 일시간 아래의 배아에서 집게로 chorion를 제거합니다. </li><li> 4 배아까지 장착을위한 슬라이드를 준비합니다. <ul><li> 직경이 약 1cm 구멍 특별히 고안된 2mm 두께의 플라스틱 슬라이드의 아래쪽에 coverslip을 봉인하기 위해 실리콘 진공 그리스를 사용합니다. </li><li> 0.7 % 아가로 오스와 슬라이드에 구멍을 채우기하고, 약 20 분 또는 강화된 때까지 대기하게 </li><li> 아가로 오스가 확정되면, 각각의 배아를 탑재하는 원통형 구멍을 형성 200μl 피펫 팁을 사용하여 작은 플러그를 제거합니다. </li></ul></li><li> 해부 현미경 아가로 오스 - 채워진 슬라이드에서 배아를 놓습니다. 배아를 마취하기 전에 tricaine의 50μl (웨스 터, 1995)를 추가합니다. </li><li> 기본 coverslip에 대한 뇌 또는 관심 영역으로 아가로 오스의 원통형 구멍에있는 배아를 위치 집게를 사용하십시오. </li><li> 실리콘 진공 그리스와 보안 다른 coverslip으로 아가로 오스의 배아를 커버. </li><li> 이미지를 사용하여 배아 거꾸로, 형광등, 레이저 - 스캔 또는 디스크 공촛점 현미경을 회전. 63x 또는 neuroepithelium 내에서 단일 세포의 고해상도 이미지를 수집하기 위해 높은에서 이미지. </li><li> 이미지는 LSM 소프트웨어에서 TIFF 파일 등 수출과 포토샵을 사용하여 분석하고 있습니다. </li></ol> 4. 대표 결과 / 결과 midbrain 및 hindbrain의 심실 사이에 24 HPF의 zebrafish의 neuroepithelium 대표 공촛점 이미지는 그림 1에 표시됩니다. 각 세포는 세포막 - 바운드 GFP와 함께 표시됩니다. <img src="/files/ftp_upload/1217/Graeden JoVE figure 1.png" alt="" width="450" height="449" /> 그림 1. memGFP - 주입 24 HPF의 zebrafish 배아의 공촛점 이미지를 살고 있습니다. GFP에 의해 명시된 각 셀에있는 24 HPF의 배아의 (A) Neuroepithelium. 지역 몇 군데는 midbrain - hindbrain 경계를 형성, midbrain 및 hindbrain의 심실 (M, H 각각)을 분리합니다.

<ol>
	<li>Gutzman, J. H., Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> Mech Dev. 125 (974), 11-12 (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish. , (1995).</li></ol>

이 비디오에서는, 우리는 단세포 zebrafish의 배아에 mRNA 분사하는 방법을 보여줍니다. 여기서는 각 셀에 레이블을 막 타겟 GFP를 인코딩 mRNA를 사용합니다. 그러면 단일 셀 해상도에서 개발 두뇌를 마운트 및 이미지하는 방법을 보여줍니다. 이 기술은 우리가 midbrain - hindbrain 경계의 형성 (구츠맨 외, 2008)에 필요한 세포 모양의 변화의 소설 유형, 기초 수축을 공부하고있었습니다. 다른 현상과 마찬가?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SeaKem GTG agarose</td>
<td> Reagent</td>
<td>Lonza</td>
<td>50027</td>
<td>&nbsp;</td>
<tr>
<td>3-aminobenzoic acid ethyl ester (Tricaine)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Capillary tubing</td>
<td>Tool</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>Borosil 1.0mmOD x 0.5mm ID/fiber 100mm</td>
</tr>
<tr>
<td>Silicone vacuum grease</td>
<td>Reagent</td>
<td>VWR</td>
<td>W0S717</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11232-20</td>
<td>Dumont #5 Bio Inox</td>
</tr>
<tr>
<td>1-200 &#x03BC;l Pipette tips</td>
<td>Tool</td>
<td>Tip One, US Scientific</td>
<td>111-0806</td>
<td>These are the correct size for 24 hpf embryos. </td>
</tr>
<tr>
<td>mMessage mMachine transcription kit</td>
<td>Reagent</td>
<td>Ambion</td>
<td>AM1340</td>
<td>SP6 RNA polymerase</td>
</tr>
<tr>
<td>Zeiss LSM 510 scanning confocal </td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>Tool</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>Tool</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PLI-100</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>Tool</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

live imaging of the zebrafish embryonic brain by confocal microscopy

이 비디오에서는, 우리는 우리의 실험실은 개발 zebrafish의 두뇌 (구츠맨 외, 2008)를 구부, 접이식하는 데 필요한 세포 모양 변경 및 rearrangements을 분석하기 위해 개발한 방법을 보여줍니다. 이러한 분석은 척추 두뇌의 3D 구조의 개발에 필요한 기본 세포 생물학의 새로운 이해를 내실수하고, 크게 신경 튜브 morphogenesis를 공부하는 능력을 증가시킵니다. 배아 zebrafish 두뇌는 neuroepithelium가 급증 내에 심실로 18시간 포스트 수정 (HPF)에서 시작 모양이다. 24 HPF으로 신경 튜브 morphogenesis의 초기 단계를 완료합니다. 방법을 사용하면 여기에 설명 한 세포 단계에서 배아는 mRNA 인코딩 막 타겟 녹색 형광 단백질 (memGFP)로 주입됩니다. 주입과 부화, 배아, 18 사이의 24 HPF 후, 아가로 오스에 거꾸로, 장착하고 공촛점 현미경으로 몇 군데. 특히, zebrafish 배아는 형광 이미징을위한 이상적인 시스템을 만들어 투명합니다. 우리의 분석은 midbrain - hindbrain 경계와 hindbrain에 초점을 가지고 있지만,이 방법은 80-100 μm의 깊이에 zebrafish에서 지역의 분석을 위해 연장 수 있습니다.

Watch this Scientific Journal Video about Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy at JoVE.com

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

이 비디오에서는, 우리는 공촛점 현미경으로 단세포 해상도 라이브 zebrafish의 배아에서 개발 척추 두뇌를 분석하는 방법을 보여줍니다. 이것은 우리가 단일 셀 zebrafish 배아를 주입하고 이후 개발 두뇌를 마운트와 이미지하는 방법을 포함합니다.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

live-imaging-of-the-zebrafish-embryonic-brain-by-confocal-microscopy

Research

JoVE Journal

Biology

18.5K 조회수.  MIT - Massachusetts Institute of Technology. 이 비디오에서는, 우리는 공촛점 현미경으로 단세포 해상도 라이브 zebrafish의 배아에서 개발 척추 두뇌를 분석하는 방법을 보여줍니다. 이것은 우리가 단일 셀 zebrafish 배아를 주입하고 이후 개발 두뇌를 마운트와 이미지하는 방법을 포함합니다.

비디오: Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

라이브 zebrafish의 배아에서 두 광자의 axotomy하고 시간이 경과 공촛점 영상

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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생물학

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

라이브 영상 Drosophila melanogaster 배아 Hemocyte 마이 그 레이션

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

이미징 및 배아 제브라 피쉬의 뇌 혈관 구조의 3 차원 재구성

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D 공 초점 현미경을 사용하여 제브라 피쉬의 두개 안면 형태 형성 분석

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

공 초점 현미경을 사용하여 트랜 인간 배아 신장 세포의 라이브 셀 이미징 및 3D 분석 안지오텐신 수용체의 유형 (1A)의 인신 매매

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

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 ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

제브라피쉬의 뇌를 침범하는 교모세포종 세포의 미세소관 역학의 라이브 이미징

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

Autofluorescence Imaging to Evaluate Red Algae Physiology

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in full sunshine. Most rhodophytes are red, however some can appear bluish, depending on the proportion of blue and red biliproteins (phycocyanin and phycoerythrin). Different phycobiliproteins can capture light at diverse wavelengths and transmit it to chlorophyll a, which makes photosynthesis under very different light conditions possible. These pigments respond to habitat changes in light, and their autofluorescence can help to study biological processes. Using Chroothece mobilis as a model organism and the spectral lambda scan mode in a confocal microscope, the adaptation of photosynthetic pigments to different monochromatic lights was studied at the cellular level to guess the species' optimal growth conditions. The results showed that, even when the studied strain was isolated from a cave, it adapted to both dim and medium light intensities. The presented method is especially useful for studying photosynthetic organisms that do not grow or grow very slowly under laboratory conditions, which is usually the case for those living in extreme habitats.

The present protocol describes step-by-step autofluorescence imaging and evaluation of phycobiliprotein changes in red algae based on spectral analysis. This is a label-free and non-destructive method to evaluate cellular adaptation to extreme habitats, when only scarce material is available and cells grow slowly, or not at all, under laboratory conditions.

홍조류 생리학을 평가하기 위한 자가형광 이미징

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...