Name: measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis
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Description: Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and Î» Hyperspectral FRET Imaging and Analysis at JoVE.com

著者らは、H188 cAMP FRETバイオセンサーとケニー・トリン(南アラバマ大学工学部)が独自に開発したプログラミングスクリプトを実行するのに要する時間を短縮するための技術的支援を提供したキース・ジャリンク博士(オランダ癌研究所とファン・レーウェンフック先端顕微鏡センター、アムステルダム、オランダ)を認めたいと考えています。
著者らは、資金源を認めたい:アメリカ心臓協会(16PRE27130004)、国立科学財団。(1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

このプロトコルは、南アラバマ大学の制度的動物の世話と使用委員会によって承認された手順に従います.
1. イメージング用の細胞、サンプル、試薬の準備
<ol><li>ラット肺微小血管内皮細胞(PMVEC)を先に説明した35を単離する。 注:細胞は、100 mm細胞培養皿上のサウスアラバマ大学モバイル、ALの細胞培養コアによって単離され、培養されました。</li>
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FRETバイオセンサーの開発により、単一細胞における環状ヌクレオチドシグナルの測定と可視化が可能となり、細胞内シグナル伝達事象13,22,37,38を可視化する大きな約束がある。しかし、FRETバイオセンサーの使用は、多くの蛍光タンパク質ベースのFRETレポーターの低いシグナル間特性およびFRETレ?...

環状アデノシン一リン酸(cAMP)は、細胞分裂、カルシウム流入、遺伝子転写、シグナル伝達を含む主要な細胞および生理学的プロセスに関与する第二のメッセンジャーである。証拠の成長体は、シグナル特異性が達成される細胞内のcAMPコンパートメントの存在を示唆しています1,2,3,4,5,6,7.最近まで、cAMP区画化は、異なるG結合受容体アゴニスト8、9、10、11によって誘発される明確な生理学的または細胞的効果に基づいて推測された。より最近では、FRETベース蛍光イメージングプローブは、細胞12、13、14におけるcAMP信号の直接測定および観察のための新しいアプローチを提供している。
フェルスター共鳴エネルギー移動(FRET)は、分子が近接15,16の場合にドナー分子とアクセプター分子間でエネルギー移動が起こる物理的現象である。FRETベース蛍光指標の開発に伴い、この物理現象は、タンパク質相互作用17、タンパク質共局在18、Ca+2シグナル伝達19、遺伝子発現20、細胞分裂21および環状ヌクレオチドシグナル伝達を研究する生物学的応用において使用されてきた。FRET ベースの cAMP 指標は、通常、cAMP 結合ドメイン、ドナーフルオロフォアおよびアクセプターフルオロフォア22から構成されます。例えば、この方法論で使用されるH188 cAMPセンサ12,22は、Epacから得られたcAMP結合ドメインから成り、ターコイズ(ドナー)と金星(アクセプター)フルオロフォアの間に挟まれる。基礎条件(非結合)では、ターコイズとヴィーナスは、蛍光色素間でFRETが発生するような向きである。結合ドメインにcAMPを結合すると、ターコイズとヴィーナスが離れて移動するような立体構造変化が起こり、FRETが減少する。
FRETベースのイメージングアプローチは、細胞内のcAMP信号を調査し、可視化するための有望なツールを提供します。しかし、現在のFRETベースの顕微鏡イメージング技術は、多くの場合、細胞内空間的明瞭度でFRETを測定するのに十分な信号強度を達成するのに部分的にしか成功していない。これは、多くのFRETレポーターの信号強度の制限、FRET効率の変化を正確に定量するために必要な高い精度、および細胞自家蛍光23、24などの交易因子の存在を含むいくつかの要因によるものである。その結果、多くの場合、弱いSNRに悩まされているFRET画像であり、FRETにおける細胞内変化の視覚化は非常に困難になります。さらに、空間的に局在したcAMP信号の調査は、ほぼ2つの空間次元でほぼ独占的に行われ、軸方向のcAMP分布はめったに25と考えられてはなされていない。これは、SNR が低いために cAMP 勾配を 3 つの空間次元で測定および視覚化する機能が妨げられたためです。低SNRを有するFRETセンサの使用の限界を克服するために、我々は、単一細胞25、26、27でFRETを測定するためのハイパースペクトルイメージングおよび分析アプローチを実施した。
ハイパースペクトルイメージングアプローチは、衛星画像28,29に存在する地上の物体を区別するためにNASAによって開発された。これらの技術は、その後、蛍光顕微鏡観察分野30に翻訳され、いくつかの商業的共焦点顕微鏡システムがスペクトル検出器を提供する。従来の(非スペクトル)蛍光イメージングでは、サンプルはバンドパスフィルタまたはレーザーラインを使用して励起され、発光は2番目のバンドパスフィルタを使用して収集され、フッ素フォロアのピーク発光波長に合わせて選択されることがよくあります。対照的に、ハイパースペクトルイメージングアプローチは、特定の波長間隔で蛍光発光26、31、32または励起33、34のいずれかの完全なスペクトルプロファイルをサンプリングすることを求める。我々のこれまでの研究では、ハイパースペクトルイメージングおよび解析アプローチは、従来のフィルタベースのFRETイメージング技術26と比較して、細胞内のFRET信号の定量化を改善できることを示した。ここでは、4次元(x、y、z、λ)ハイパースペクトルFRETイメージングおよび解析を行い、cAMP分布を3つの空間次元で測定および可視化する方法論を提示する。これらのアプローチは、単一細胞25におけるアゴニスト誘発cAMP空間勾配の可視化を可能にした。興味深いことに、アゴニストによっては、cAMP勾配が細胞内で明らかである可能性がある。ここで提示される方法論は、FRET測定の精度を向上させるために、不均一な背景および細胞自家蛍光のスペクトルの非混合を利用する。この方法論は、cAMP FRETバイオセンサーを用いた肺微小血管内皮細胞(PMVEC)で実証されているが、この方法論は、代替FRETレポーターまたは代替細胞株で使用するために容易に変更することができる。

<table>
	<tbody>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>Invitrogen</td>
			<td>A7816</td>
			<td>Attofluor contains steel cell chambers and a rubber O-ring. Cell chamber holds the coverslip and O-ring provides a lock in mechanism to hold the buffer in cell chamber with out leakage</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td>Solvent used to prepare stock solution forskolin.</td>
		</tr>
		<tr>
			<td>DRAQ5 Fluoroscent Probe Solution</td>
			<td>Thermo Scientific</td>
			<td>62252</td>
			<td>Nuclear label</td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>Contains nutrients and growth factors for the cells to grow and divide in the culture dishes.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F6178</td>
			<td>Growth factor suppliment that is added to culture medium, DMEM</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F3917</td>
			<td>Adenyly cyclase activator.</td>
		</tr>
		<tr>
			<td>H188 Cyclic AMP FRET biosensor</td>
			<td>Netherland Cancer Institute, Dr. K. Jalink</td>
			<td>Gift</td>
			<td>Plasmid encoding Turquoise (donor fluorophore), Venus (acceptor fluorophore), and binding domain obtained from Epac.</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>image.net</td>
			<td>Free download</td>
			<td>Another image processing platform used to extact spectral information and image processing.</td>
		</tr>
		<tr>
			<td>Integrating Sphere</td>
			<td>Ocean Optics</td>
			<td>FOIS-1</td>
			<td>Used to measure illumination intensity of the laser line at different laser intensities (?).</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td>Coverslips are coated with laminin and this helps in cell attachment, growth and motility of the cell.</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Kit</td>
			<td>Invitrogen</td>
			<td>L3000-015</td>
			<td>Transfection reagent used to transfect cells with H188 FRET biosensor</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2019a</td>
			<td>Image processing operations (linear unmixing and FRET efficiency calculations) are performed by writing custom programs in MATLAB programming environment</td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon Instruments</td>
			<td>Nikon A1R</td>
			<td>Spectral image acquisition is performed using confocal microscope.</td>
		</tr>
		<tr>
			<td>Nikon Elements Software</td>
			<td>Nikon Instruments</td>
			<td>Software dongle</td>
			<td>used to export and handle nd2 image files (multidimensional image files) that are aquired using Nikon A1R</td>
		</tr>
		<tr>
			<td>NIST-Traceable Calibration Lamp</td>
			<td>Ocean Optics</td>
			<td>LS-1-CAL-INT</td>
			<td>A lamp with a known spectrum for use as a standard</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>coomonly used buffer suring cell culture</td>
		</tr>
		<tr>
			<td>Pulmonary Microvascular Endothelial Cells (PMVECs)</td>
			<td>In house - Cell culture core, Univeristy of South Alabama</td>
			<td>Isolated from Rat pulmonary microvasculature</td>
			<td>PMVECs form inner lining of a blood vessel.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/ml)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>antibiotics are added to culture medum to prevent contamination of the cells.</td>
		</tr>
		<tr>
			<td>Pre-Cleaned Gold Seal Micro Slides</td>
			<td>Clay Adams</td>
			<td>3010</td>
			<td>Microscope slides used for cell fixation</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mounting Media</td>
			<td>Invitrogen</td>
			<td>P36961</td>
			<td>If samples are fixed using antifade mountant, then the later protects fluoroscent dyes and chromophores from fading.</td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics</td>
			<td>QE65000</td>
			<td>Used to measure spectral response of the light source (?)</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Digests the protein-protein bond between the cell and cell matrix and helps to disscociate and lift the cells during cell plating.</td>
		</tr>
		<tr>
			<td>Tyrodes Buffer</td>
			<td>Made in-house</td>
			<td>Made in-house</td>
			<td>Tyrodes buffer is used to make working solutions and to maintain cells in aqueous solution during image acquisition.</td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Laminin coated coverslips are placed in 6-well culture dish (one coverlisps/well). Cells along with medium is added into each well.</td>
		</tr>
		<tr>
			<td>25 mm Round Microscope Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12545102</td>
			<td>Cells were grown on round glass coverslips</td>
		</tr>
		<tr>
			<td>60X Ojective</td>
			<td>Nikon Instruments</td>
			<td>Plan Apo VC 60X/1.2 WI &#8734;/0.15-0.18 WD 0.27</td>
			<td>water immersion and commonly used objective for cells</td>
		</tr>
	</tbody>
</table>

measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis

サイクリックAMPは、細胞および生理活性の広い範囲に関与している第二のメッセンジャーです。いくつかの研究は、cAMP信号が区画化され、区画化がcAMPシグナル経路内のシグナル特異性に寄与することを示唆している。Fӧrster共鳴エネルギー伝達(FRET)ベースのバイオセンサーの開発は、細胞内のcAMP信号を測定し、可視化する能力を高めています。ただし、これらの測定値は、多くの場合、2 つの空間次元に限定され、データの誤解釈につながる可能性があります。これまで、3空間次元(x、y、z)でのcAMP信号の測定は非常に限られており、本質的に低信号対雑音比(SNR)を示すFRETセンサの使用に関する技術的な制限によりあります。さらに、従来のフィルタベースのイメージング手法は、スペクトルクロストーク、限定的な信号強度、自己蛍光などのさまざまな要因により、局在化した細胞下領域でcAMP信号を正確に測定するのには効果的でないことがよくあります。これらの制限を克服し、FRETベースのバイオセンサーを複数の蛍光ホルで使用できるようにするために、FRET効率を計算するためのスペクトル特異性と、FRET信号を同時交流自発および/または追加の蛍光標識からのシグナルからスペクトル的に分離する能力を提供するハイパースペクトルFRETイメージングおよび分析アプローチを開発しました。ここでは、ハイパースペクトルFRETイメージングを実装するための方法論と、スペクトルアンミックスを行うためにアンダーサンプリングもオーバーサンプリングもしない適切なスペクトルライブラリを構築する必要性を提示する。この方法論は、肺微小血管内皮細胞(PMVEC)における3次元cAMP分布の測定に用いるが、この方法論は、細胞タイプの範囲におけるcAMPの空間分布を研究するために使用することができる。

このプロトコルは、ハイパースペクトルFRETイメージングの使用と、生細胞における3つの空間次元におけるcAMP勾配を測定するための分析アプローチについて説明する。これらの結果の生成には、データの分析と定量を行う際に細心の注意が必要な重要なステップがいくつかあります。これらの主要なステップには、適切なスペクトルライブラリの構築、バックグラウンドスペクトルの非混合?...

Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and Î» Hyperspectral FRET Imaging and Analysis at JoVE.com

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and &lambda; Hyperspectral FRET Imaging and Analysis

Fӧrster共鳴エネルギー伝達(FRET)ベースのセンサの信号対雑音比(SNR)が低いため、cAMP信号の測定は特に3つの空間次元において困難でした。ここでは、3つの空間次元でcAMP分布を測定できるハイパースペクトルFRETイメージングおよび解析方法論について述べる。

4次元(x、y、z、λ)ハイパースペクトルFRETイメージングと解析を用いた生体細胞における3次元cAMP分布の測定

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are ...

This protocol is significant as it provides a method for measuring and visualizing FRET or cyclic AMP signals in three spatial dimensions in individual cells. Cyclic AMP distributions may occur axially in addition to laterally and it is important to acquire FRET cyclic AMP image data in three dimensions to sample these distributions. On the day of imaging, warm Tyrodes buffer to 37 degrees Celsius in a water bath and mount a cover slip seated with the transfected cells of interest into a cell chamber.Secure the top of the chamber with a mounting gasket to prevent leaking and use a delicate task wiper to remove any excess medium or adherent cells from the bottom of the cover slip. Add 800 microliters of working buffer and four microliters of five millimolar nuclear label to the cell chamber and gently rock the chamber for five to 10 seconds. Then cover the cell chamber with aluminum foil for a 10 minute incubation at room temperature.To image the cells, select the 60X water immersion objective on a confocal microscope equipped with a spectral detector and add a drop of water to the objective. Place the loaded cell chamber onto the microscope stage and tune the filter knob to select the appropriate filter set. Select the fluorescence widefield mode and use the eyepieces to select a field of view containing cells expressing the FRET cyclic AMP sensor.Open the imaging software, select the confocal mode, and unlock the laser interlock button. Open the A1 settings menu. Check the boxes corresponding to the 405 and 561 nanometer laser lines and set these spectral detections to SD, the resolution to 10, and the channels to 32.Select the start and end wavelength values to set the wavelength range. Open the A1 Settings menu to select the binning skip icon. Select the number 15 box and click OK.Set the laser intensities to eight percent for the 405 nanometer laser and to two percent for the 561 nanometer laser.Set the detector gain to 149 and the pinhole radius to 2.4 Airy disk units. Set the scan speed to 25 spectral frames per second, and select the unidirectional scan direction. Set the count to four and the scan size to 1024 by 1024.To define the z-stack acquisition parameters, click View, Acquisition Control, and ND Acquisition, and enter the file destination and name in the pop window to save the ND file. Click the z-series box and click Live in the A1 Plus Settings window to open a live viewing window. Adjust the focus knob to focus on the cells while looking in the preview window.Adjust the focus knob on the microscope to select the top of the cell and click Top in the ND Acquisition window to set the current position as the top. Adjust the focus knob on the microscope to select the bottom of the cell and click Bottom to set the current position as the bottom. Enter one micrometer for the step size.Select Top Bottom for the z-scan direction and click run to acquire a z-stack. When the z-stack acquisition is complete, use a pipette to gently add the reagent of interest to the chamber without disturbing the cells. After 10 minutes, change the file name and acquire a second z-stack image.After imaging, create new folders with the same file names as the spectral z-stack images and open the spectral image file of interest. Click File, Import/Export, and Export ND Document. In the pop-up window, select the newly created folder, TIFF for the file type, Mono image for each channel, and keep bit depth.Click export to export the individual files to the folder and open the linear spectral unmixing software. Open the Linear Unmixing. m file and click Run.Browse and select the folder containing the exported TIFF file sequence and click OK to open the wavelength and z-slice window. Copy the file name of the first file without the z-slice and channel number from the folder and paste it into the first step of the Enter of the imagename box, enter the number of channels in the Enter the number of wavelength bands box, and the number of z-slices in the Enter the number of Z Slices box and click OK.Then in the pop-up window, browse and select the Wavelength. mat file and click Open.Browse and select the Library. mat file in the new pop-up window and click Open again to initiate the slice unmixing. To calculate the FRET efficiency, open the multiFRRCF.m programming script and enter the number of experimental trials to analyze in the How many folders to reslice box. Click OK.Browse to select the unmixed folders and click OK again. In the new pop-up window, set the scaling factor to 12.4, the threshold to 56, the X, Y, and Z frequency to five, five, and one respectively, and the smoothing algorithm to Gaussian.Then click Run to perform the FRET measurements and reslicing. To map the FRET efficiency to cyclic AMP levels, open the Mapping_FRET.Efficiency_to_cAMP_concentration. m file and click Run.Navigate to and select the first grayscale FRET image and click OK.Then open the FRET cyclic AMP images to inspect the distribution of the cyclic AMP signals in three dimensions. In these images, three-dimensional views of false colored raw hyperspectral image data acquired using confocal microscopy at baseline and 10 minutes after forskolin treatment can be observed. In this analysis, an acceptor fluorophore was completely photo destructed allowing spectral signatures of the donor and acceptor with one-to-one stoichiometry to be obtained.Non-transfected cells labeled with a nuclear dye were utilized to obtain the pure spectrum of the dye fluorophore. Combining the spectra of the donor, acceptor, and nuclear dye fluorophores allows the creation of a three component library. Here, the sources of three background spectral signatures can be observed.Although these signals are distributed non-uniformly within the sample and cannot simply be subtracted out, adding the spectral signatures of these signals to the spectral library and using linear unmixing to separate the signals provides an approach for removing these confounding signals from the donor and acceptor signals prior to calculating their FRET efficiencies. The six component spectral library can then be used to perform linear spectral unmixing for each slice in the axial image. Here, the changes in FRET efficiency and cyclic AMP levels in different XY plane slices can be observed allowing a comparison between the baseline conditions and the conditions 10 minutes after forskolin treatment.This technique could also be used to quantify the FRET efficiency of other reporters in three dimensions in both cells and tissues.

Research

JoVE Journal

Biology

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral FRET Imaging and Analysis

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

生物学

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

セルフメイドサイトスピン装置を用いてヒト多能性幹細胞の免疫細胞化学的解析

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

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Imaging Cell Shape Change in Living Drosophila Embryos

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

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Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

共焦点顕微鏡を用いた生細胞におけるエンドサイトーシスおよび細胞質区画における同時pH測定

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

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

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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) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

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Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

FRETベースの共焦点イメージングを使用した培養平滑筋細胞の小胞体内カルシウム変動の測定

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

FRET Imaging in Three-dimensional Hydrogels

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...