この作業はホープScholarの賞の防衛時代の部（第W81XWH - 05から0396まで）とエドワードB.ブラウンIIIに医歯薬学総合研究賞のPewの学者によって賄われていた。

1。光学系の位置を合わせます。 MP - FRAP実験のための重要な設備は、次のとおりです。モード同期レーザ光源、ポッケルスセル（光変調用）、パルスジェネレータ、ダイクロイック、対物レンズ、蛍光発光フィルター、ゲート光電子増倍管、およびデータ記録システム（光子計数器をとマルチチャンネルスケーラ）。 2。安全モニターの電源を決定します。 <ol><li>サンプル内で蛍光を生成するための、低い、しかし合理的なパワーにレーザーを減衰させる。 </li><li>サンプル内にフォーカス。 </li><li>はるかに長い予想される蛍光の回復よりも時間スケールを介して統合するフォトンカウンタを設定します。焦点体積が試料内の固定された（私達の顕微鏡のソフトウェアでこれはポイントスキャンによって達成される）と、フォトンカウントデータの合理的な数を記録。 </li><li>電力を増加させると光子のカウントデータの別のシリーズを取る。フォトンカウントの増加が電力の増加を基準にして大幅に減少すると表示されるまで繰り返します。 </li><li>ログ（電力）の関数としてプロットログ（光子計数）。二つの傾きからの偏差は、モニター中に漂白示します。お使いのモニタの電源として、以下のこのカットオフですが、これはノイズ比に対する合理的な信号を可能にする電源を選択してください。 </li></ol> 3。パルス発生器とマルチチャンネルスケーラに入力パラメータを決定する。 <ol><li>拡散係数と回復時間の文献に基づいて、及び/またはStokes - Einsteinの方程式および拡散方程式の一般向けのエンベロープの推定値を決定する。 </li><li>パルス発生器とスケーラ上で必要なパラメータ値を設定します。 <ol class="lalpha"><li>パルスジェネレータでは、事前に漂白剤や漂白剤回数を設定します。経験則の良いセットは、事前に漂白剤が予想される半分の回復時間に比べ1-2倍大きくなるはず、と漂白時間は十分の一、期待半分の回復時間でなければならないということです。 </li><li>スケーラーで、約半分漂白剤の持続時間にビンの幅を設定し、記録ごとのビンの数を設定して、選択されたビンの幅と、レコードごとのビンの製品がより大きいか、予想される完全な回復に加えて、事前にほぼ等しくなるように - 漂白剤や漂白剤の回。 </li><li>パルス発生器に戻ると、ビンの幅の倍の製品レコードあたりのビンの数の逆数よりちょうど小さい値に等しいpre-bleach/bleach/monitorシーケンスの頻度を設定します。 * </li><li>最後に、レコード数を設定/選択したモニターの電源での信号強度に基づいて、スケーラーにスキャン。 *これは、パルス発生器で設定した完全なpre-bleach/bleach/monitorシーケンス（1/frequency）のための時間は、スケーラに設定されたデータ収集時間（ビン幅×ビン/記録）を超えていることを非常に重要です。この基準が満たされていない場合は、複数の漂白剤のトリガーは、スケーラの単一のレコード内に表示されることがあります。 </li></ol></li><li>以前に決定された許容レベルにモニターの電源を設定します。モニター漂白テスト中に決定される漂白カット上記で200mW程度に漂白剤のパワーを設定します。これらの力は、PCの結晶間の異なる電圧などのセットの両方があります。 </li><li>顕微鏡をシール、ライトを遮断し、PMTをオンにして、パルスジェネレータとスケーラを開始する、顕微鏡のソフトウェア上のポイントのスキャンを開始します。 </li><li> 1）事前に漂白剤蛍光、2）蛍光直後の漂白剤、およびデータセットの最後に3）蛍光：曲線をプロットし、3つの重要なポイントをマークすることで、その結果回復曲線を分析する。蛍光値を使用して前と直後の漂白剤よりハーフリカバリ時間を推定する。 </li><li>ハーフ回復時間のこの見積もりで、新しいプレ漂白剤、漂白剤の値、およびビンの幅の期間を決定する前に概説した親指のルールを使用してください。また、より完全な回復を達成するために必要な時間を見積もるために、データセットの最後にプリ漂白剤蛍光と蛍光を使用してください。経験則として、データはデータの後半のために報告されるように完全な回復を可能にする期間のために収集する必要があります。 </li><li>あなたのカーブが強い漂白剤とスムーズな回復を示すまで、パルスジェネレータとスケーラ上でパラメータを調整、パラメータを新しいカーブを取り、調整を続ける。可能であれば、漂白剤の持続時間ができることに留意してください、そして、親指のルールの下に小さくする必要があります。浅すぎる、または深すぎる、漂白剤が達成されている場合は漂白剤のパワーをも調整することができます。 </li></ol> 4。励起の飽和のためのテスト。 <ol><li>以前に決定された適切なモニターと漂白剤の力を、使用して、MP - FRAP曲線の一連を取る。適切な数学的形態と非線形最小二乗フィッティングアルゴリズム（以下、&quot;データ解析&quot;を参照）を使用して、事前に漂白剤蛍光に対して正規化各回復を、分析する。漂白剤の深さのパラメータの近似値を記録します。</li><li>漂白剤のパワーの値を増加させることMP - FRAPカーブの連続するシリーズを取り、分析を続行。 </li><li>ログ（電力）の関数としてプロットログ（漂白剤の深さのパラメータ）。二つの斜面から逸脱すると、励起の飽和を示している。強力な漂白剤が得られるが、飽和が発生しない漂白パワーを選択してください。 </li></ol> 5。 MP - FRAP曲線の服用を続け。 <ol><li>上記のように、すべてのパラメータが正しく設定される、曲線の服用を続け。 </li></ol> 6。データ解析。 <ol><li>蛍光データから事前に漂白剤や漂白剤、データなど時間のデータを設定し、調整を取り外しますその直後の漂白剤蛍光値はt = 0に対応。 </li><li>平均的なプレ漂白剤蛍光に関して生じる蛍光の回復を正規化する。 </li><li>適切な数学モデルやMATLABなどのlsqcurvefitは関数として非線形、最小二乗フィッティングアルゴリズムを使用して、正規化曲線をフィット。ここで紹介するモデルは、拡散と対流の影響を受けて蛍光回復を占めている。 <img src="/files/ftp_upload/1636/1636eq.jpg" alt="式" /> F oは事前に漂白剤の平均蛍光ここで、βは、漂白剤の深さのパラメータである、τDは特徴的な拡散性の回復時間は、Rは半径方向（W R）幅の軸方向の二乗の比率（w Z）です。二光子焦点ボリューム、τVX = W R / V X、τVY = W R / V Y、τVZ = W のz / V Z、およびV 2 = V × 2 + V Y 2 + V Z 2である対流の速度。結像面に限局フローの場合、τVZ→∞と1 /τVX + 1 /τVYは、1 /τVRに置き換えることができます。対流がない場合には、分子の両方の指数が1に移動し、式が大幅にのみ2つのフィッティングパラメータ、β、τ、Dに、低減されます。拡散係数は、簡単にD = W R 2 /8τDとして計算されます。 </li></ol> 7。代表的な結果。 <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="図1" /> 図1代表的な漂白剤とFITC - BSAのための回復曲線。良好な回復曲線は、データの後半のために報告されている完全な回復での蛍光で、強力な漂白剤とスムーズな回復を示す。 <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="図2" /> 図2。FITC - BSA（赤）と関連付けられている最小二乗フィット（黒）のための正規化された回復曲線。このフィットは、文献と一致52.9 UM2 / sの拡散係数を、もたらします。

<ol>
	<li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Biophys J. 60, 73-76 (1990).</li><li>Brown, E. B., Wu, E. S., Zipfel, W., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+molecular+diffusion+in+solution+by+multiphoton+fluorescence+photobleaching+recovery.">Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.</a> Biophys J. 77, 2837-2849 (1999).</li><li>Sullivan, K. D., Sipprell, W. H. B. r. o. w. n., Jr, E. B., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+model+of+fluorescence+recovery+expands+the+application+of+multiphoton+fluorescence+recover+after+photobleaching+in+vivo.">Improved model of fluorescence recovery expands the application of multiphoton fluorescence recover after photobleaching in vivo.</a> Biophys J. 96, 5082-5094 (2009).</li></ol>

光退色後の多光子蛍光回復の力は、3次元分解能で厚さのサンプルを調査する能力にあります。 1990年代の開発以来、MP - FRAPは細胞体、 生体外で厚い組織切片、 および in vivo組織と間質への拡散係数を（または類似のトランスポートパラメータ）を決定するために使用されています。この記事では、MP - FRAP実験、ならびに、実験パラメータの設定データを取得し、回復曲線...

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<td>Ti:sapphire laser</td>
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<td>Short pass dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma Technologies</td>
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<td>Photomultiplier tube</td>
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measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

光退色後のマルチ蛍光回復は、高分子の拡散係数を（または類似のトランスポートパラメータ）を測定するために使用する顕微鏡技術である、との両方に適用することができます。<em&gt; in vitroで</em&gt;と<em&gt; in vivoで</em&gt;生物系。光退色後のマルチ蛍光回復は、ビームを減衰し、photobleached分子を置換するための関心のびまん性の領域の外側から、まだ、蛍光分子などの蛍光をモニターする、強力なレーザーフラッシュを使用して、蛍光試料内に関心領域を退色によって実行されます。我々は、ポッケルスセル（レーザー変調器）を介して、サンプルへのレーザースキャンボックスと対物レンズを介して光路に沿ってレーザービームを調整することで、私たちのデモを開始します。簡単にするために、我々は、水性蛍光色素のサンプルを使用します。私たちは、その後、適切な実験を含めて我々のサンプルのパラメータ、モニターや漂白力、漂白剤の持続時間、ビン幅（フォトンカウンティング用）、および蛍光回復時間を決定します。次に、我々は、回復曲線、主に強化されたスループットは、LabVIEW（ナショナルインスツルメンツ、オースティン、テキサス州）を経由して自動化できるプロセスをとるための手順を説明します。最後に、拡散係数は、MATLAB（Mathworks社、ネイティック、マサチューセッツ州）などのソフトウェアを使用して容易にプログラム可能な最小自乗法のアルゴリズムを使用して、適切な数学モデルにリカバリデータをフィッティングによって決定されます。

Watch this Scientific Journal Video about Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching at JoVE.com

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

この記事では、光退色後の多光子蛍光の回復を使用して拡散係数を測定するための手順を説明します。我々は、サンプルへの光路に沿ってレーザーを合わせ、適切な実験パラメータを決定することによって開始されます、その後の生成を続行し、最終的にフィッティング蛍光回復曲線。

光退色後二光子蛍光回復を経由して拡散係数の測定

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) ...

measuring-diffusion-coefficients-via-two-photon-fluorescence-recovery

Research

JoVE Journal

Biology

11.1K ビュー.  University of Rochester. この記事では、光退色後の多光子蛍光の回復を使用して拡散係数を測定するための手順を説明します。我々は、サンプルへの光路に沿ってレーザーを合わせ、適切な実験パラメータを決定することによって開始されます、その後の生成を続行し、最終的にフィッティング蛍光回復曲線。

ビデオ: Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

ライブゼブラフィッシュ胚における二光子ベースの光活性化

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

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.

スペクトル的に解決された二光子顕微鏡を用いたGタンパク質共役受容体相互作用の in vivo定量で

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

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

胚性幹細胞を生体内でクロマチン蛋白質のダイナミクスを測定するアッセイ（FRAP＆FLIP）を光退色

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

複数のターゲットトレース（MTT）による原形質膜中の分子拡散のマッピング

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I ·クライオキットを使用してマウスの精子の凍結保存と回復

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

k空間画像相関分光法を用いて、EGFPタグ付きプラズマ膜タンパク質の拡散係数を簡単に測定

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

細胞内カルシウムの二光子イメージング<sup&gt; 2+</sup&gt;取扱い及び内皮における一酸化窒素産生および単離されたラット大動脈の平滑筋細胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...