この作品は、国立眼研究所グラント1 R15 EY13649 - 01、でとカナダ自然科学工学研究評議会（NSERC）から個々の発見の助成金によって支えられている。金属加工用、およびデバイスの設計と建設に関する協議のための徳達ガレスに感謝。

<ol>
	<li>Goldreich, D. &. a. m. p. ;. a. m. p., Kanics, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+acuity+is+enhanced+in+blindness.">Tactile acuity is enhanced in blindness.</a> J. Neurosci. 23, 3439-3445 (2003).</li><li>Goldreich, D. &. a. m. p. ;. a. m. p., Kanics, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+blind+and+sighted+humans+on+a+tactile+grating+detection+task.">Performance of blind and sighted humans on a tactile grating detection task.</a> Percept. Psychophys. 68, 1363-1371 (2006).</li><li>Kontsevich, L. L. &. a. m. p. ;. a. m. p., Tyler, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+adaptive+estimation+of+psychometric+slope+and+threshold.">Bayesian adaptive estimation of psychometric slope and threshold.</a> Vision. Res. 39, 2729-2737 (1999).</li><li>Bleyenheuft, Y., Cols, C., Arnould, C. &. a. m. p. ;. a. m. p., Thonnard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+tactile+spatial+resolution+from+6+to+16+years.">Age-related changes in tactile spatial resolution from 6 to 16 years.</a> Somatosens. Mot. Res. 23, 83-87 (2006).</li><li>Johnson, K. O. &. a. m. p. ;. a. m. p., Phillips, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+spatial+resolution.+I.+Two-point+discrimination,+gap+detection,+grating+resolution,+and+letter+recognition.">Tactile spatial resolution. I. Two-point discrimination, gap detection, grating resolution, and letter recognition.</a> J. Neurophysiol. 46, 1177-1192 (1981).</li><li>Gibson, G. O. &. a. m. p. ;. a. m. p., Craig, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+force+and+conformance+on+tactile+intensive+and+spatial+sensitivity.">The effect of force and conformance on tactile intensive and spatial sensitivity.</a> Exp. Brain Res. 170, 172-181 (2006).</li><li>Johnson, K. O. &. a. m. p. ;. a. m. p., Phillips, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotating+drum+stimulator+for+scanning+embossed+patterns+and+textures+across+the+skin.">A rotating drum stimulator for scanning embossed patterns and textures across the skin.</a> J. Neurosci. Methods. 22, 221-231 (1988).</li><li>Craig, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grating+orientation+as+a+measure+of+tactile+spatial+acuity.">Grating orientation as a measure of tactile spatial acuity.</a> Somatosens. Mot. Res. 16, 197-206 (1999).</li><li>Killebrew, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dense+array+stimulator+to+generate+arbitrary+spatio-temporal+tactile+stimuli.">A dense array stimulator to generate arbitrary spatio-temporal tactile stimuli.</a> J. Neurosci. Methods. 161, 62-74 (2007).</li></ol>

空間的に構造化された機械的刺激の制御された送達は、市販の機器（コンピュータ画面、オーディオスピーカー）を使用することのできる視覚または聴覚刺激、の配信に直面していない課題ポーズ。このような理由から、多くの触覚心理物理実験は依然として手動刺激の配信を使用して行われます。 触覚刺激のマニュアル配信は時間がかかる、と研究者の一部に細心の注意と集中力を必要とします。皮膚に対して垂直の変形の約1〜2 mmの手動アプリケーションが使用された&quot;こと例えば、Bleyenheuftら4レポート...審査官は、皮膚と対策を歪める可能性が格子の間の任意のせん断応力を避けるために特に気配りだった。 ..&quot;手動の刺激の配達は比較的遅いと集中集約的であるなどの理由で、この方法を使用して多くの研究は、はい/いいえ（トライアルごとに1つの刺激）ではなく、二区間強制選択プロトコルを採用していない。 残念なことに、細心の注意が取られている場合でも、手動の刺激の配信は、多くの刺激のパラメータが制御されていないまま。これらのパラメータは、刺激の力、発症の速度、期間、および皮膚における刺激の表面の安定性が含まれています。これらのパラメータのうち、おそらくパフォーマンス上の影響力に関して最も研究は、刺激の力です。人間は、500から1200ミクロン5からのインデントの深さの変化に応じて指先で格子の方向弁別における漸進的な改善を示し、それ以上の改善は50グラムと200グラム6の間観察されないものの、パフォーマンスは、50グラムフォース1よりも10グラムの力で優れています。このような格子の検出（溝加工からスムースを区別する）などの他のアプリケーションの場合は、力の増加は有意に10〜200グラム2,6の範囲全体に渡ってパフォーマンスを向上させる。 手動のテストに関連するこれらの課題を克服するために、我々は、自動化システム、TAPSを開発した。建物のTAPSの私たちの目的は、制御された触覚刺激アプリケーションのための、シンプルで安全な、汎用性、効率的かつ手頃な価格のデバイスを作成することでした。重力の制御を使用するには、触覚刺激を適用する簡単で安全な方法です。皮膚に対する力は重力に起因することを超えることができないので安全性は、確保されている。強力なリニアアクチュエータは、皮膚の上に重力の力を転送するし、連絡先を撤回するのに役立つ。それはあらゆる刺激の面を収容するようにデバイスは、汎用性のある半イン​​チ径ロッドの両端（または上に接続されている）に加工し、心理物理学のさまざまなプロトコルを実行するようにプログラムすることができます。 TAPSは、それが急速に刺激を展開すると、効率的であり、適応アルゴリズムを時間節約実行することができます。最後に、TAPSのコンポーネントは、小さな助成金の保有者に手頃な価格です。モータは、約1500ドルの費用が、電気部品（コンピュータを除くが、PCIボードとステッピングモータドライバを含む）、約6000ドル。ミニフライス盤および付属品は約1,000ドル購入す​​ることができる、または専門の機械工は、工場への刺激のピースを雇用し、その他の小さな部品を作ることができます。 我々は、このシステムは他のユーザーによって複製されることを期待、そして近年では進展5,7,8,9を有望見ている制御された触覚のテスト、に向けた動きを促進する役割を果たす。

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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>Delrin plastic rods 0.5&#x201D; diameter 3&#x201D; long </td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>ZRD-08</td>
<td>Rods from which the stimulus pieces are made</td>
</tr>
<tr>
<td>Chrome-steel bearing, 0.5" bore diameter</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>BR-08-01</td>
<td>Pivot for the rotating rod</td>
</tr>
<tr>
<td>Plastic bearing, 0.5&#x201D; bore, 1.125&#x201D; outer diameter, 0.25&#x201D; thick</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>BRP-08-01</td>
<td>Wheel at end of rotating rod</td>
</tr>
<tr>
<td>Fiberglass curtain, 0.75&#x201D; thick</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>9781T83</td>
<td>Sound absorbing curtain around lower table</td>
</tr>
<tr>
<td>Silicone O-rings 5/16" inside diameter</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>AS568A- 011</td>
<td>To reduce vibration of stimulus pieces</td>
</tr>
<tr>
<td>Anti-Vibration Pads 2"x 2", 93 PSI max </td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>60105K61</td>
<td>Put under the linear actuator</td>
</tr>
<tr>
<td>Rubber sandwich mounts 1/2" H x 3/8" W </td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>9378K11</td>
<td>Put under linear actuator base</td>
</tr>
<tr>
<td>Stepper motor Nema 23, 3-stack </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>S23</td>
<td>For rotating the stimulus disk</td>
</tr>
<tr>
<td>Linear rodless actuator</td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>R2S23N-105A-18-l-M35M</td>
<td>To control rotating bar movement</td>
</tr>
<tr>
<td>1 Hall effect switch &#x201C;normally open&#x201D; config. </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>RP1</td>
<td>Actuator home sensor</td>
</tr>
<tr>
<td>2 Hall effect switches &#x201C;normally closed&#x201D; config. </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>RP2</td>
<td>Actuator limit sensors</td>
</tr>
<tr>
<td>Micro switch</td>
<td>&nbsp;</td>
<td>Honeywell</td>
<td>FSG15N1A</td>
<td>Finger force sensor</td>
</tr>
<tr>
<td>Retro-reflective sensor</td>
<td>&nbsp;</td>
<td>Honeywell</td>
<td>FE7B-RB6VG-M</td>
<td>Piece position sensor</td>
</tr>
<tr>
<td>Motor controller board, closed-loop control</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-step-4CX</td>
<td>Mediates communication between computer and Nudrive</td>
</tr>
<tr>
<td>Nudrive stepper motor driver</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Nudrive 4SX-211</td>
<td>Sends command voltages to the linear actuator and disk stepper motors</td>
</tr>
<tr>
<td>Data acquisition board</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-MIO-16E-1</td>
<td>Reads finger force sensor</td>
</tr>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>&nbsp;</td>
<td>Programming language</td>
</tr>
<tr>
<td>Valuemotion library</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>&nbsp;</td>
<td>Motor control routines</td>
</tr>
<tr>
<td>Photoelectric sensor</td>
<td>&nbsp;</td>
<td>Panasonic</td>
<td>PM-K53-C1</td>
<td>Disk home sensor</td>
</tr>
<tr>
<td>Mini milling machine</td>
<td>&nbsp;</td>
<td>Sherline</td>
<td>2010-DRO</td>
<td>For machining the stimulus surfaces</td>
</tr>
<tr>
<td>Ohaus precision mass set</td>
<td>&nbsp;</td>
<td>Edmund Scientific</td>
<td>&nbsp;</td>
<td>Mass on rotating bar</td>
</tr>
<tr>
<td>Parrish Magic Line aluminum cake pan bottom, 9&#x201D; diam.</td>
<td>&nbsp;</td>
<td>Sur La Table</td>
<td>&nbsp;</td>
<td>Rotating disk machined with square cut-outs to hold the stimulus pieces</td>
</tr>		</tbody>
		</table>
		</div>

a tactile automated passive-finger stimulator (taps)

触覚空間視力テストは神経科学の研究と臨床評価の両方で使用されていますが、いくつかの自動化されたデバイスは、皮膚に制御する空間的に構造化刺激を提供するために存在する。その結果、捜査官はしばしば手動で触覚刺激を適用する。手動の刺激のアプリケーションは時間がかかる、捜査官の一部に細心の注意と集中力を必要とし、多くの刺激のパラメータが制御されていないまま。ここでは、コンピュータ制御の触覚刺激システム、発症の速度、接触力、接触の持続時間をコントロール、肌に空間的に構造化された刺激を適用する触覚自動パッシブフィンガー刺激（TAPS）を、記述する。 TAPSは、効率的に心理物理学的手続きの様々なを行うことができる多目的な、プログラム可能なシステムです。我々は、TAPSのコンポーネントを説明し、TAPSは、2つの間隔強制選択の触覚格子の向きのテストを管理するために使用される方法を示します。 対応する著者：ダニエルGoldreich

Watch this Scientific Journal Video about A Tactile Automated Passive-Finger Stimulator TAPS at JoVE.com

A Tactile Automated Passive-Finger Stimulator TAPS

触覚自動パッシブフィンガースティミュレーター（TAPS）：我々は、タッチの感覚を調査するためのコンピュータ制御装置を説明します。我々は、TAPSのコンポーネントを説明し、TAPSは、2つの間隔強制選択の触覚格子の向きのテストを管理するために使用される方法を示します。

触覚自動パッシブフィンガースティミュレーター（TAPS）

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering ...

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Biology

A Tactile Automated Passive-Finger Stimulator (TAPS)

13.6K ビュー.  Duquesne University. 触覚自動パッシブフィンガースティミュレーター（TAPS）：我々は、タッチの感覚を調査するためのコンピュータ制御装置を説明します。我々は、TAPSのコンポーネントを説明し、TAPSは、2つの間隔強制選択の触覚格子の向きのテストを管理するために使用される方法を示します。

ビデオ: A Tactile Automated Passive-Finger Stimulator TAPS

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

スモールハーツの半自動光ビート解析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

生物学

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

自動プロテオーム処理のためのデジタルマイクロ流体

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ：半自動化された葉の形状計測のためのImageJのプラグイン

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

ゼブラフィッシュ胚における組織特異的蛍光の半自動イメージング

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

ダイナミックカルシウムの自動分析<sup&gt; 2 +</supイメージシーケンス内の&gt;の信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

自動化された蛍光寿命イメージング顕微鏡を利用してオープンソースのハイコンテンツな解析

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

自動イメージングシステムを用いたiPS細胞の合流点の測定

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...