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

Författarna vill erkänna Dr. Kees Jalink (Nederländerna Cancer Institute och van Leeuwenhoek Center for Advanced Microscopy, Amsterdam, Nederländerna) för att ge oss H188 cAMP FRET biosensor och Kenny Trinh (College of Engineering, University of South Alabama) för teknisk hjälp för att minska den tid det tar att köra våra specialutvecklade programmeringsskript.
Författarna vill erkänna finansieringskällorna: American Heart Association (16PRE27130004), National Science Foundation; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

Detta protokoll följer förfaranden som godkänts av University of South Alabama Institutional Animal Care and Use Committee.
1. Cell-, prov- och reagenspreparat för avbildning
<ol><li>Isolera råtta pulmonell microvascular endotelceller (PMVECs) som beskrivits tidigare35. OBS: Celler isolerades och odlades av Cell Culture Core vid University of South Alabama, Mobile, AL på 100 mm cellkulturrätter.</li>
	<li>Fröisolerade PMVECs på 25 mm runda glaslock och lå...

<ol>
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Utvecklingen av FRET-biosensorer har gjort det möjligt att mäta och visualisera cykliska nukleotidsignaler i enstaka celler, och det finns ett stort löfte om att visualisera subcellulära signalhändelser13,22,37,38. Användningen av FRET-biosensorer innebär dock flera begränsningar, inklusive de låga signal-till-brus-egenskaperna hos många fluorescerande proteinbaserade FRET-reportrar o...

Cykliskt adenosinmonofofat (cAMP) är en andra budbärare som är involverad i viktiga cellulära och fysiologiska processer inklusive celldelning, kalciumtillströmning, gentranskribering och signaltransduktion. En växande mängd bevis tyder på förekomsten av cAMP-fack i cellen genom vilken signalspecifikitet uppnås1,2,3,4,5,6,7. Fram till nyligen, cAMP compartmentalization härleddes baserat på distinkta fysiologiska eller cellulära effekter framkallas av olika G-kopplade receptor agonister8,9,10,11. På senare tid har FRET-baserade fluorescensavbildningssonder gett nya metoder för direkt mätning och observation av cAMP-signaler i en cell12,13,14.
Förster resonans energiöverföring (FRET) är ett fysiskt fenomen där energiöverföring sker mellan donator- och acceptormolekyler på ett icke-radiativt sätt när molekylerna är i närheten15,16. Med utvecklingen av FRET-baserade fluorescerande indikatorer har detta fysiska fenomen använts i biologiska tillämpningar för att studera proteinproteininteraktioner17, protein samlokalisering18, Ca+2 signalering19, genuttryck20, celldelning21 och cyklisk nukleotid signalering. FRET-baserade cAMP-indikatorer består vanligtvis av en cAMP-bindande domän, en donatorfluorofor och en acceptorfluorfor22. H188 cAMP-sensorn12,22 somanvänds i denna metod består till exempel av en cAMP-bindningsdomän som erhållits från Epac, inklämd mellan turkos (donator) och Venus (acceptor) fluorforer. Vid basala förhållanden (obundna) är turkos och Venus i en orientering så att FRET uppstår mellan fluorforerna. Vid bindning av cAMP till bindningsdomänen sker en konformationsförändring så att turkos och Venus rör sig isär vilket resulterar i en minskning av FRET.
FRET-baserade bildmetoder erbjuder ett lovande verktyg för att undersöka och visualisera cAMP-signaler i en cell. Nuvarande FRET-baserade mikroskopiska bildframställning tekniker är dock ofta bara delvis framgångsrika för att uppnå tillräcklig signal styrka för att mäta FRET med subcellulära rumsliga klarhet. Detta beror på flera faktorer, inklusive den begränsade signalstyrkan hos många FRET-reportrar, den höga precisionsnivå som krävs för att exakt kvantifiera förändringar i FRET-effektivitet och närvaron av förvirrande faktorer, såsom cellulär autofluorescens23,24. Resultatet är ofta en FRET-bild som plågas av svag SNR, vilket gör visualisering av subcellulära förändringar i FRET mycket svårt. Dessutom har undersökning av rumsligt lokaliserade cAMP-signaler nästan uteslutande utförts i endast två rumsliga dimensioner och den axiella cAMP-fördelningen har sällan ansettsvara 25. Detta beror sannolikt på att låg SNR hindrade förmågan att mäta och visualisera cAMP-gradienter i tre rumsliga dimensioner. För att övervinna begränsningarna med att använda FRET-sensorer med låg SNR har vi implementerat hyperspektrala avbildnings- och analysmetoder för att mäta FRET i enstakaceller 25,26,27.
Hyperspektrala avbildningsmetoder utvecklades av NASA för att skilja markbundna objekt som finns i satellitbilder28,29. Dessa tekniker har sedan dess översatts till fluorescensmikroskopifältet30, med flera kommersiella konfokala mikroskopsystem som erbjuder spektraldetektorer. I traditionell (icke-spektral) fluorescensavbildning är provet upphetsad med hjälp av ett bandpassfilter eller en laserlinje, och utsläppet samlas in med hjälp av ett andra bandpassfilter, ofta valt för att matcha fluorforens topputsläppsvåglängd. Däremot försöker hyperspektrala avbildningsmetoder att prova en komplett spektralprofil av antingen fluorescensutsläppet26,31,32 eller excitation33,34 med specifika våglängdsintervall. I våra tidigare studier visade vi att hyperspektral avbildning och analysmetoder kan erbjuda förbättrad kvantifiering av FRET-signaler i celler jämfört med traditionella filterbaserade FRET-bildtekniker26. Här presenterar vi en metodik för att utföra 4-dimensionella (x, y, z och λ) hyperspektrala FRET-avbildning och analys för att mäta och visualisera cAMP-fördelningar i tre rumsliga dimensioner. Dessa metoder har tillåtit visualisering av agonist-inducerad cAMP rumsliga gradienter i enstaka celler25. Intressant, beroende på agonisten, cAMP gradienter kan vara uppenbart i celler. Den metod som presenteras här använder spektral unmixing av icke-enhetlig bakgrund och cellulära autofluorescens för att förbättra noggrannheten i FRET mätningar. Medan denna metod visas i pulmonell microvascular endotelceller (PMVECs) med hjälp av en cAMP FRET biosensor, metoden kan lätt ändras för användning med alternativa FRET reportrar eller alternativa cellinjer.

<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

Cyclic AMP är en andra budbärare som är involverad i ett brett spektrum av cellulära och fysiologiska aktiviteter. Flera studier tyder på att cAMP signaler är fackindela, och att compartmentalization bidrar till att signalera specificitet inom cAMP signalering utbildningsavsnittet. Utvecklingen av Fӧrster resonansenergiöverföring (FRET) baserade biosensorer har främjat förmågan att mäta och visualisera cAMP-signaler i celler. Dessa mätningar är dock ofta begränsade till två rumsliga dimensioner, vilket kan leda till feltolkning av data. Hittills har det endast gjorts mycket begränsade mätningar av cAMP-signaler i tre rumsliga dimensioner (x, y och z), på grund av de tekniska begränsningarna vid användning av FRET-sensorer som i sig uppvisar låg signal-till-brusförhållande (SNR). Dessutom är traditionella filterbaserade avbildningsmetoder ofta ineffektiva för noggrann mätning av cAMP-signaler i lokaliserade subcellulära regioner på grund av en rad faktorer, inklusive spektralkors, begränsad signalstyrka och autofluorescens. För att övervinna dessa begränsningar och tillåta FRET-baserade biosensorer att användas med flera fluorforer har vi utvecklat hyperspektrala FRET-avbildnings- och analysmetoder som ger spektralspecifikitet för beräkning av FRET-effektivitet och förmågan att spektroally separera FRET-signaler från förvirrande autofluorescens och / eller signaler från ytterligare fluorescerande etiketter. Här presenterar vi metodiken för att implementera hyperspektrala FRET-avbildningar samt behovet av att konstruera ett lämpligt spektralbibliotek som varken är undersamplat eller översamplat för att utföra spektral-unmixing. Medan vi presenterar denna metod för mätning av tredimensionella cAMP fördelningar i pulmonell microvascular endotelceller (PMVECs), denna metodik kan användas för att studera rumsliga fördelningar av cAMP i en rad celltyper.

Detta protokoll beskriver användningen av hyperspektrala FRET imaging och analys metoder för att mäta cAMP gradienter i tre rumsliga dimensioner i levande celler. Det finns flera viktiga steg involverade i att generera dessa resultat, för vilka noggrann uppmärksamhet krävs när man analyserar och kvantifierar data. Dessa viktiga steg inkluderar konstruktion av ett lämpligt spektralbibliotek, bakgrundsspektralt unmixing, tröskelvärden för att identifiera cellgränser och FRET-effektivitetsberäkningar. <strong c...

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

På grund av inneboende låg signal-till-brus-förhållande (SNR) av Fӧrster resonansenergiöverföringsbaserade sensorer (FRET) har mätningen av cAMP-signaler varit utmanande, särskilt i tre rumsliga dimensioner. Här beskriver vi en hyperspektral FRET imaging och analys metodik som möjliggör mätning av cAMP fördelning i tre rumsliga dimensioner.

Mätning av 3-dimensionella cAMP-fördelningar i levande celler med 4-dimensionell (x, y, z och λ) hyperspektrala FRET-avbildningar och analyser

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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