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

作者感谢Kees Jalink博士（荷兰癌症研究所和荷兰阿姆斯特丹范利乌文霍克高级显微镜中心）为我们提供了H188 cAMP FRET生物传感器和肯尼·特里恩（南阿拉巴马大学工程学院）的技术帮助，以减少运行我们自定义开发的编程脚本所花的时间。
作者要感谢资金来源：美国心脏协会（16PRE27130004），国家科学基金会：（1725937） NIH， S100D020149， S10RR027535， R01HL058506， P01HL066299）.）

该协议遵循南阿拉巴马大学机构动物护理和使用委员会批准的程序。
1. 细胞、样品和试剂成像准备
<ol><li>孤立大鼠肺微血管内皮细胞（PMVECs），如前所述35。 注：细胞被隔离和培养的细胞培养核心在南阿拉巴马大学，移动，AL在100毫米细胞培养菜。</li>
	<li>种子分离PMVECs在25毫米圆形玻璃盖片上，并让他们在37°C的孵化器中生长，直到细胞达到至少80%的汇?...

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FRET生物传感器的研制使单细胞环核苷酸信号的测量和可视化，对13、22、37、38等亚细胞信号事件的可视化大有可为。然而，使用FRET生物传感器存在一些局限性，包括许多荧光蛋白基FRET记者的低信号到噪声特性，以及FRET记者的弱透射或表达效率（这在某些细胞系中可能特别具有挑战性，如PMVECs）23、24...

环腺苷单磷酸盐（cAMP）是参与关键细胞和生理过程的第二个信使，包括细胞分裂、钙的涌入、基因转录和信号转导。越来越多的证据表明，细胞中存在cAMP隔间，通过这些隔间可以达到1、2、3、4、5、6、7的信号特异性。直到最近，CAMP分隔断是根据不同的G耦合受体激动剂8，9，10，11诱导的独特的生理或细胞效应推断的。最近，基于FRET的荧光成像探测器为直接测量和观察12、13、14细胞中的CAMP信号提供了新的方法。
Fürster共振能量转移（FRET）是一种物理现象，当分子接近15、16时，能量转移以非辐射的方式发生在供体和接受分子之间。随着基于FRET的荧光指标的发展，这种物理现象已用于生物应用，研究蛋白质-蛋白质相互作用17，蛋白质共定位18，Ca+2信号19，基因表达20，细胞分裂21和环核苷酸信号。基于FRET的cAMP指标通常包括一个CAMP结合域，一个捐赠者氟和一个接受荧光22。例如，H188 cAMP 传感器12，22用于此方法包括从 Epac 获得的 cAMP 结合域，夹在绿松石（捐赠者）和金星（接受者）荧光之间。在基础条件下（未绑定），绿松石和金星处于一个方向，因此FRET发生在萤管之间。将 cAMP 与绑定域结合后，会发生构象变化，使绿松石和金星分开，导致 FRET 减少。
基于 FRET 的成像方法为研究细胞内的 cAMP 信号提供了一个很有前途的工具。然而，目前的基于FRET的微观成像技术往往只部分成功达到足够的信号强度，以测量具有亚细胞空间清晰度的FRET。这是由于几个因素，包括许多FRET记者的信号强度有限，准确量化FRET效率变化所需的高精度，以及存在混淆因素，如细胞自发光23，24。结果往往是受弱 SNR 困扰的 FRET 图像，使得 FRET 的子细胞变化的可视化变得非常困难。此外，空间定位 CAMP 信号的调查几乎只在两个空间维度中执行，轴向 CAMP 分布很少被视为25。这可能是因为低 SNR 妨碍了在三个空间维度中测量和可视化 cAMP 梯度的能力。为了克服使用低SNR的FRET传感器的限制，我们采用了高光谱成像和分析方法，在单个细胞25、26、27中测量FRET。
美国宇航局开发了超光谱成像方法，以区分卫星图像28、29中存在的地球物体。此后，这些技术被翻译成荧光显微镜场30，几个商业共聚焦显微镜系统提供光谱探测器。在传统的（非光谱）荧光成像中，样品使用带通滤镜或激光线激发，并且使用第二个带通滤波器收集发射，通常选择以匹配荧光的峰值发射波长。相比之下，高光谱成像方法寻求以特定波长间隔对荧光发射26、31、32或激发33、34的完整光谱剖面进行采样。在我们之前的研究中，我们发现，与传统的基于过滤器的FRET成像技术相比，高光谱成像和分析方法可以改善细胞中FRET信号的量化。在这里，我们提出了一种执行 4 维（x、y、z 和 +）高光谱 FRET 成像和分析的方法，以测量和可视化三个空间维度的 cAMP 分布。这些方法使单细胞25中激动剂诱导的CAMP空间梯度可视化。有趣的是，根据激动剂的不同，CAMP梯度在细胞中可能很明显。此处提供的方法利用不均匀背景和细胞自发光的光谱解密，以提高 FRET 测量的准确性。虽然这种方法在使用 cAMP FRET 生物传感器的肺微血管内皮细胞 （PMVECs） 中得到了证明，但该方法可以很容易地修改，用于替代 FRET 记者或替代细胞系。

<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

循环AMP是第二个信使，参与广泛的细胞和生理活动。一些研究表明，cAMP 信号是分隔的，隔间化有助于 cAMP 信号通路中的信号特异性。基于Fürster共振能量转移（FRET）的生物传感器的发展，进一步增强了测量和可视化细胞中CAMP信号的能力。但是，这些测量通常局限于两个空间维度，这可能导致对数据的误解。迄今为止，由于使用本身信号与噪声比 （SNR） 较低的 FRET 传感器的技术限制，在三个空间维度（x、y 和 z）中对 CAMP 信号的测量非常有限。此外，由于光谱相声、信号强度有限和自发光等一系列因素，传统的基于滤波器的成像方法通常对精确测量局部子细胞区域的 CAMP 信号无效。为了克服这些限制，并允许基于FRET的生物传感器与多个荧光素一起使用，我们开发了高光谱FRET成像和分析方法，为计算FRET效率提供光谱特异性，并能够将FRET信号与混淆的自发光和/或来自其他荧光标签的信号隔离开来。在这里，我们介绍了实施高光谱FRET成像的方法，以及需要构建一个适当的光谱库，既不采样不足，也不采样过采，以执行光谱未混合。虽然我们提出了测量肺微血管内皮细胞（PMVECs）中三维CAMP分布的方法，但这种方法可用于研究CAMP在一系列细胞类型的空间分布。

本协议描述了使用高光谱 FRET 成像和分析方法测量活细胞中三个空间维度的 cAMP 梯度。生成这些结果涉及几个关键步骤，在分析和量化数据时需要仔细注意这些步骤。这些关键步骤包括构建适当的光谱库、背景光谱未混合、识别细胞边框的阈值以及 FRET 效率计算。 图1 说明了测量活细胞中FRET效率和cAMP水平所涉及的所有步骤的示意图流。如果执行得当，这些成像和分析步骤?...

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

由于基于 Fürster 共振能量转移 （FRET） 传感器的固有的低信号与噪声比 （SNR），CAMP 信号的测量一直具有挑战性，尤其是在三个空间维度方面。在这里，我们描述了一个高光谱FRET成像和分析方法，允许测量三个空间尺寸的CAMP分布。

使用 4 维（x、y、z 和 +）高光谱 FRET 成像和分析测量活细胞中的三维 cAMP 分布

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

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

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

科研

生物学

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

人多能干细胞的免疫组织化学分析，使用自制Cytospin器械

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

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

影像生活细胞的形状变化果蝇胚胎

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

在毛发再生的上皮 - 间充质信号的快速遗传分析

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

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

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

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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

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

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

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

荧光成像与一纳米精度（FIONA）

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

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

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

FRET成像的三维水凝胶

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 ...