第一Siveラボでこのテクニックを開発した博士ジェニファーGutzmanに感謝します。親切にコードするプラスミドCAAX - GFPのmRNAを提供Dana - Farber癌研究所ボストン、マサチューセッツ州でもJBグリーンのおかげで。共焦点イメージングは​​、ホワイトヘッドでWMケック財団生体イメージング施設で行われた。 HSはNIHMH70926によってサポートされています。 EGは、NSFの事前博士フェローシップでサポートされています。

1。インジェクションのmRNAの準備<ol><li>この手順で使用されているmRNAがコードするプラスミドCAAX - EGFP（memGFP）mRNAから転写される。最初Gutzmanら、2008によるとプラスミドを線形化する。 </li><li>その後mMessage mMachineキットを使用してmemGFP mRNAを転写。 </li><li> -801μg/μl、分注し、およびストアに得られたmRNAを希釈℃を</li><li>一つの細胞の段階で胚の幅は、レーンと1％アガロースの射出成形金型を準備します。 </li><li>注射の前日に、女性から男性を分離交尾ケージを設定します。 </li><li>注射の日に、注射の準備をマイクロピペットプラーを使用してキャピラリー針を引き抜きます。 </li></ol> 2。 mRNAを注入<ol><li>注射の日に、氷上に1μg/μlmemGFPのmRNAのアリコートを解凍。 </li><li> 200ng/μlの最終濃度に水でmRNAが1:5に希釈し、氷上に置きます。 </li><li> 200ng/μlで準備memGFPのmRNAの1μlのでキャピラリー針をロードする。 </li><li>ガソリンインジェクターに接続されているマイクロマニピュレータにロードされた針を置きます。 </li><li> 1nlに注入量を調整します。 </li><li>前日から交配のケージに設定野生型の魚で仕切りを引っ張る。 </li><li>とすぐにそれらが配置されるように胚を収集する。オリエントそれら離れてマイクロマニピュレータの側の単一細胞で胚の培地（ウェスター、1995）でカバーアガロースの射出成形金型インチ</li><li>絨毛膜とmRNAは単一のセルに直接堆積されるように、卵黄を介して注入する。実験ごとに約50の胚を注入する。セルが分割されている場合に注入しないでください。 </li><li> 28℃で一晩胚の培地でインキュベートする胚</li></ol> 3。マウントおよびイメージング胚<ol><li>胚をマウントし、イメージに、興味のある時点の前に実体顕微鏡一時間で胚から鉗子で絨毛膜を除去。 </li><li> 4胚まで取り付けるためのスライドを準備します。 <ul><li>直径約1cm穴を内蔵した特別設計の2ミリメートルの厚さのプラスチック製のスライドの下側にカバースリップを密封するために、シリコーン真空グリースを使用してください。 </li><li> 0.7％アガロースでスライドの穴を埋める、と約20分または硬化するまで放置</li><li>アガロースが固化した後、それぞれの胚をマウントするに円筒状の穴を形成するために、200μlのピペットチップを使用して、小さなプラグを取り外します。 </li></ul></li><li>解剖顕微鏡下でアガロースを充填したスライド上に胚を置きます。胚を麻酔tricaine50μlの（ウェスター、1995）を追加します。 </li><li>基本的なカバースリップに対する脳や関心領域とアガロースの円筒形の穴に胚を配置するために鉗子を使用してください。 </li><li>シリコン真空グリースで保護された別のカバースリップとアガロースの胚をカバーする。 </li><li>反転、蛍光灯、レーザー走査型または回転するディスク共焦点顕微鏡を用いて画像胚を。神経上皮内の単一セルの高解像度の画像を収集するために63x以上で画像。 </li><li>画像は、LSMソフトウェアからのTIFFファイルとしてエクスポートし、Photoshopを使用して分析している。 </li></ol> 4。代表的な結果/成果図1に示すように、中脳と後脳の心室の間に24 HPFのゼブラフィッシュの神経上皮の代表的な共焦点画像です。各セルは、膜結合型GFPで標識されている。 <img src="/files/ftp_upload/1217/Graeden JoVE figure 1.png" alt="" width="450" height="449" /> 図1。 memGFP注入24 HPFのゼブラフィッシュ胚の共焦点イメージングを生きる。 GFPによる概説、各セルで24 HPFの胚の（A）神経上皮。地域撮像さは中脳後脳境界を形成し、中脳と後脳の脳室（M、Hはそれぞれ）を分離。

<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>

このビデオでは、我々は単一細胞のゼブラフィッシュの胚へのmRNAの注入のための方法を示しています。ここでは、各セルにラベルを付けるために細胞膜をターゲットとしたGFPをコードするmRNAを使用してください。我々は、単一セルの分解能で脳の発達をマウントし、イメージする方法を示しています。このテクニックは、私たちは中脳後脳境界（Gutzmanら、2008）の形成に必要な細胞の形状変...

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<td>SeaKem GTG agarose</td>
<td> Reagent</td>
<td>Lonza</td>
<td>50027</td>
<td>&nbsp;</td>
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<td>3-aminobenzoic acid ethyl ester (Tricaine)</td>
<td>Reagent</td>
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<td>A-5040</td>
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<td>Capillary tubing</td>
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<td>Frederick Haer and Co.</td>
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<td>1-200 &#x03BC;l Pipette tips</td>
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<td>Zeiss LSM 510 scanning confocal </td>
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<td>&nbsp;</td>
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<td>Tool</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PLI-100</td>
<td>&nbsp;</td>
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<td>Micropipette puller</td>
<td>Tool</td>
<td>Sutter Instruments</td>
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live imaging of the zebrafish embryonic brain by confocal microscopy

このビデオでは、我々の研究室が開発してゼブラフィッシュの脳（Gutzmanら、2008）曲げると折るために必要な細胞の形状の変更と再配置を分析するために開発した方法を示しています。このような分析は、脊椎動物の脳の三次元構造の開発に必要な基本的な細胞生物学の新たな理解物を得る、と大幅に神経管の形態形成を研究する我々の能力を向上させます。胚ゼブラフィッシュの脳は、神経上皮が膨らま内の心室として、18時間後に受精（HPF）で始まる形をしている。 24 HPFによって、神経管の形態形成の最初のステップは完了です。方法ここで説明を使用して、1細胞段階における胚をコードするmRNAの細胞膜をターゲットとする緑色蛍光タンパク質（memGFP）を注入されています。注射やインキュベーション、胚、現在18〜24 HPF後、アガロースで、反転、マウントされ、共焦点顕微鏡で撮像。特に、ゼブラフィッシュの胚は、その蛍光イメージングのための理想的なシステムとなって透過的です。我々の分析は、中脳後脳境界と後脳に焦点を当てているが、この方法は80〜100μmの深さにゼブラフィッシュで任意の領域の分析のために拡張することができる。

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

このビデオでは、我々は共焦点顕微鏡による単一細胞の解像度でのライブゼブラフィッシュの胚で開発脊椎動物の脳を分析するためにする方法を示しています。これは、我々は、単一セルのゼブラフィッシュの胚を注入し、その後、脳の発達をマウントし、イメージする方法が含まれています。

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

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 ビュー.  MIT - Massachusetts Institute of Technology. このビデオでは、我々は共焦点顕微鏡による単一細胞の解像度でのライブゼブラフィッシュの胚で開発脊椎動物の脳を分析するためにする方法を示しています。これは、我々は、単一セルのゼブラフィッシュの胚を注入し、その後、脳の発達をマウントし、イメージする方法が含まれています。

ビデオ: 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.

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

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

生物学

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.

のライブイメージングキイロショウジョウバエ胚性血球移行

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.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

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.

イメージングおよび胚ゼブラフィッシュにおける脳血管構造物の3次元再構成

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共焦点顕微鏡を用いたゼブラフィッシュにおける顔面形態形成を分析する

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.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

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.

共焦点顕微鏡を使用してトランスフェクトされたヒト胚性腎細胞におけるアンジオテンシン受容体1a型の人身売買のライブセルイメージングと3D解析

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.

高圧急速凍結、マイクロ波を用いたコントラスト強調、およびボリューム イメージングのために埋め込む最小限の樹脂による生体試料作製

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.

ゼブラフィッシュ脳に侵入する膠芽腫細胞における微小管動態のライブイメージング

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.

紅藻生理機能を評価するための自己蛍光イメージング

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