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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Os autores gostariam de reconhecer o Dr. Kees Jalink (Instituto de Câncer dos Países Baixos e o Centro de Microscopia Avançada van Leeuwenhoek, Amsterdã, Holanda) por nos fornecer o biosensor H188 cAMP FRET e Kenny Trinh (College of Engineering, University of South Alabama) para ajuda técnica na redução do tempo de tempo que leva para executar nossos scripts de programação desenvolvidos sob medida.
Os autores gostariam de reconhecer as fontes de financiamento: American Heart Association (16PRE27130004), National Science Foundation; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

Este protocolo segue os procedimentos aprovados pelo Comitê Institucional de Cuidados e Uso de Animais da Universidade do Sul do Alabama.
1. Preparação de células, amostras e reagentes para imagem
<ol><li>Isolar as células endoteliais microvasculares pulmonares de ratos (PMVECs) como descrito anteriormente35. NOTA: As células foram isoladas e cultivadas pelo Núcleo de Cultura Celular da Universidade do Sul do Alabama, Mobile, AL em pratos de cultura celular ...

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O desenvolvimento de biosensores FRET permitiu a medição e visualização de sinais nucleotídeos cíclicos em células únicas, e há grande promessa de visualização de eventos de sinalização subcelular13,22,37,38. No entanto, o uso de biosensores FRET apresenta várias limitações, incluindo as características de baixo sinal-ruído de muitos repórteres fret à base de proteína fluor...

Monofosfato de adenosina cíclica (cAMP) é um segundo mensageiro envolvido em processos celulares e fisiológicos chave, incluindo divisão celular, influxo de cálcio, transcrição genética e transdução de sinais. Um conjunto crescente de evidências sugere a existência de compartimentos cAMP na célula através dos quais a especificidade de sinalização é alcançada1,2,3,4,5,6,7. Até recentemente, a compartimentação cAMP era inferida com base em efeitos fisiológicos ou celulares distintos induzidos por diferentes agonistas receptores acopladosG 8,9,10,11. Mais recentemente, as sondas de imagem de fluorescência baseadas em FRET forneceram novas abordagens para a medição direta e observação de sinais cAMP em uma célula12,13,14.
A transferência de energia de ressonância förster (FRET) é um fenômeno físico no qual ocorre a transferência de energia entre moléculas doadoras e aceitadoras de forma não radiativa quando as moléculas estão próximas15,16. Com o desenvolvimento de indicadores fluorescentes baseados em FRET, esse fenômeno físico tem sido utilizado em aplicações biológicas para estudar interações proteína-proteína17, co-localização proteica18, Ca+2 sinalizando19, expressão genética20,divisão celular21 e sinalização nucleotídea cíclica. Os indicadores cAMP baseados em FRET normalmente consistem em um domínio de vinculação cAMP, um fluoróforo doador e um fluoróforoaceitor 22. Por exemplo, o sensor H188 cAMP12,22 utilizado nesta metodologia consiste em um domínio de ligação cAMP obtido da Epac, sanduíche entre fluoroforos turquesa (doador) e Vênus (aceitador). Em condições basais (sem entrada), Turquesa e Vênus estão em uma orientação de tal forma que o FRET ocorre entre os fluoroforos. Ao vincular o cAMP ao domínio de ligação, ocorre uma alteração conformacional de tal forma que Turquesa e Vênus se afastam, resultando em uma diminuição do FRET.
As abordagens de imagem baseadas em FRET oferecem uma ferramenta promissora para investigar e visualizar sinais cAMP dentro de uma célula. No entanto, as técnicas atuais de imagem microscópica baseadas em FRET são muitas vezes apenas parcialmente bem sucedidas em alcançar força de sinal suficiente para medir o FRET com clareza espacial subcelular. Isso se deve a vários fatores, incluindo a intensidade limitada do sinal de muitos repórteres da FRET, o alto nível de precisão necessário para quantificar com precisão as mudanças na eficiência do FRET e a presença de fatores de confusão, como a autofluorescência celular23,24. O resultado é muitas vezes uma imagem FRET que é atormentada por SNR fraco, tornando muito difícil a visualização de mudanças subcelulares no FRET. Além disso, a investigação de sinais cAMP espacialmente localizados tem sido realizada quase exclusivamente em apenas duas dimensões espaciais e a distribuição axial cAMP raramente foi considerada25. Isso é provável porque o SNR baixo impediu a capacidade de medir e visualizar gradientes cAMP em três dimensões espaciais. Para superar as limitações do uso de sensores FRET com baixo SNR, implementamos abordagens hiperespectrais de imagem e análise para medir o FRET em células únicas25,26,27.
Abordagens de imagem hiperespectral foram desenvolvidas pela NASA para diferenciar objetos terrestres presentes em imagens de satélite28,29. Essas técnicas foram traduzidas para o campo de microscopia de fluorescência30,com vários sistemas comerciais de microscópio confocal oferecendo detectores espectrais. Em imagens tradicionais (não espectrais) de fluorescência, a amostra é animada usando um filtro de passe de banda ou uma linha laser, e a emissão é coletada usando um segundo filtro de passe de banda, muitas vezes selecionado para corresponder ao comprimento de onda de emissão máxima do fluoróforo(s). Em contrapartida, as abordagens de imagem hiperespectral buscam amostrar um perfil espectral completo da emissão de fluorescência26,31,32 ou excitação33,34 em intervalos específicos de comprimento de onda. Em nossos estudos anteriores, mostramos que as abordagens de imagem e análise hiperespectral podem oferecer uma quantificação melhorada dos sinais DE TRAC nas células quando comparadas às técnicas tradicionais de imagem FRET baseadas emfiltros 26. Aqui, apresentamos uma metodologia para a realização de imagens e análises hiperespectrais de FRET hiperdimensionais (x, y, z e λ) para medir e visualizar distribuições cAMP em três dimensões espaciais. Essas abordagens permitiram a visualização de gradientes espaciais cAMP induzidos por agonista em célulasúnicas 25. Curiosamente, dependendo do agonista, gradientes cAMP podem ser aparentes nas células. A metodologia aqui apresentada utiliza o descompreamento espectral de fundo não uniforme e autofluorescência celular para melhorar a precisão das medições de FRET. Embora essa metodologia seja demonstrada em células endoteliais microvasculares pulmonares (PMVECs) utilizando um biosensor CAMP FRET, a metodologia poderia ser facilmente modificada para uso com repórteres fret alternativos ou linhas celulares alternativas.

<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

O AMP cíclico é um segundo mensageiro que está envolvido em uma ampla gama de atividades celulares e fisiológicas. Vários estudos sugerem que os sinais de cAMP são compartimentados, e que a compartimentação contribui para a sinalização da especificidade dentro da via de sinalização cAMP. O desenvolvimento de biosensores baseados em transferência de energia de ressonância Fӧrster (FRET) promoveram a capacidade de medir e visualizar sinais cAMP nas células. No entanto, essas medidas são muitas vezes limitadas a duas dimensões espaciais, o que pode resultar em má interpretação dos dados. Até o momento, houve apenas medições muito limitadas de sinais cAMP em três dimensões espaciais (x, y e z), devido às limitações técnicas no uso de sensores FRET que inerentemente exibem baixa relação sinal/ruído (SNR). Além disso, as abordagens tradicionais de imagem baseadas em filtros são muitas vezes ineficazes para a medição precisa de sinais cAMP em regiões subcelulares localizadas devido a uma série de fatores, incluindo crosstalk espectral, força limitada do sinal e autofluorescência. Para superar essas limitações e permitir que os biosensores baseados em FRET sejam usados com fluoroforos múltiplos, desenvolvemos abordagens hiperespectrais de imagem e análise de FRET que fornecem especificidade espectral para calcular a eficiência do FRET e a capacidade de separar espectralmente os sinais FRET de confundir autofluorescência e/ou sinais de rótulos fluorescentes adicionais. Aqui, apresentamos a metodologia de implementação de imagens FRET hiperespectrais, bem como a necessidade de construir uma biblioteca espectral apropriada que não seja subamostra nem supersamplada para realizar descomprturações espectrais. Enquanto apresentamos essa metodologia para medição de distribuições tridimensionais de cAMP em células endoteliais microvasculares pulmonares (PMVECs), essa metodologia poderia ser usada para estudar distribuições espaciais de cAMP em uma gama de tipos celulares.

Este protocolo descreve o uso de abordagens hiperespectrais de imagem e análise de FRET para medir gradientes cAMP em três dimensões espaciais em células vivas. Existem várias etapas fundamentais envolvidas na geração desses resultados, para as quais é necessária uma atenção cuidadosa ao analisar e quantificar os dados. Essas etapas-chave incluem a construção de uma biblioteca espectral apropriada, descompramento espectral de fundo, limiar para identificar bordas celulares e cálculos de eficiência do FRET....

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

Devido à relação sinal-ruído inerente (SNR) de sensores baseados em transferência de energia de ressonância (FRET) Fӧrster, a medição dos sinais cAMP tem sido desafiadora, especialmente em três dimensões espaciais. Aqui, descrevemos uma metodologia hiperespectral de imagem e análise de FRET que permite a medição da distribuição cAMP em três dimensões espaciais.

Medição de distribuições 3-dimensionales cAMP em células vivas usando imagens e análises hiperespectrais de FRET 4-Dimensional (x, y, z e λ) Hiperespectrais

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

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Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

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Medição simultânea em pH endocítica e citossólicos Compartimentos em células vivas usando microscopia confocal

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

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