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

Forfatterne vil gjerne anerkjenne Dr. Kees Jalink (The Netherlands Cancer Institute og van Leeuwenhoek Center for Advanced Microscopy, Amsterdam, Nederland) for å gi oss H188 cAMP FRET biosensor og Kenny Trinh (College of Engineering, University of South Alabama) for teknisk hjelp til å redusere tiden det tar å kjøre våre spesialutviklede programmeringsskript.
Forfatterne ønsker å anerkjenne finansieringskildene: American Heart Association (16PRE27130004), National Science Foundation; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

Denne protokollen følger prosedyrer godkjent av University of South Alabama Institutional Animal Care and Use Committee.
1. Celle-, prøve- og reagensforberedelse for avbildning
<ol><li>Isoler rotte lungemikrovaskulære endotelceller (PMVEC) som beskrevet tidligere35. MERK: Celler ble isolert og dyrket av Cell Culture Core ved University of South Alabama, Mobile, AL på 100 mm cellekulturretter.</li>
	<li>Frøisolerte PMVECer på 25 mm runde glassdeksler og la dem...

<ol>
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Utviklingen av FRET biosensorer har tillatt måling og visualisering av sykliske nukleotidsignaler i enkeltceller, og det er stort løfte om å visualisere subcellulære signalhendelser13,22,37,38. Bruken av FRET biosensorer presenterer imidlertid flere begrensninger, inkludert de lave signal-til-støy-egenskapene til mange fluorescerende proteinbaserte FRET-reportere og de svake transfeksjons-...

Syklisk adenosinmonofosfat (cAMP) er en annen budbringer involvert i viktige cellulære og fysiologiske prosesser, inkludert celledeling, kalsiumtilstrømning, gentranskripsjon og signaltransduksjon. En voksende mengde bevis antyder eksistensen av cAMP-rom i cellen der signal spesifisitet oppnås1,2,3,4,5,6,7. Inntil nylig ble cAMP-oppdeling utledet basert på distinkte fysiologiske eller cellulære effekter indusert av forskjellige G-koblede reseptoragonister8,9,10,11. Mer nylig har FRET-baserte fluorescensavbildningssonder gitt nye tilnærminger for direkte måling og observasjon av cAMP-signaler i en celle12,13,14.
Förster resonans energioverføring (FRET) er et fysisk fenomen der energioverføring skjer mellom donor og akseptormolekyler på en ikke-radiativ måte når molekylene er i nærheten15,16. Med utviklingen av FRET-baserte fluorescerende indikatorer har dette fysiske fenomenet blitt brukt i biologiske applikasjoner for å studere proteinproteininteraksjoner17, proteinkolokalisering18, Ca+2 som signaliserer19, genuttrykk20, celledeling21 og syklisk nukleotidsignalering. FRET-baserte cAMP-indikatorer består vanligvis av et cAMP-bindingsdomene, en donorfluofor og en akseptorfluofor22. For eksempel består H188 cAMP-sensoren12,22 som brukes i denne metoden av et cAMP-bindingsdomene hentet fra Epac, smurt mellom turkis (donor) og Venus (akseptor) fluoroforer. Ved basale forhold (ubundet) er Turkis og Venus i en orientering slik at FRET oppstår mellom fluoroforene. Ved binding av cAMP til bindingsdomenet oppstår en konformasjonsendring slik at Turkis og Venus beveger seg fra hverandre, noe som resulterer i en reduksjon i FRET.
FRET-baserte bildemetoder tilbyr et lovende verktøy for å undersøke og visualisere CAMP-signaler i en celle. Imidlertid er nåværende FRET-baserte mikroskopiske avbildningsteknikker ofte bare delvis vellykkede med å oppnå tilstrekkelig signalstyrke til å måle FRET med subcellulær romlig klarhet. Dette skyldes flere faktorer, inkludert den begrensede signalstyrken til mange FRET-reportere, det høye presisjonsnivået som kreves for å nøyaktig kvantifisere endringer i FRET-effektivitet, og tilstedeværelsen av forvirrende faktorer, for eksempel cellulær autofluorescence23,24. Resultatet er ofte et FRET-bilde som er plaget av svak SNR, noe som gjør visualisering av subcellulære endringer i FRET svært vanskelig. I tillegg har undersøkelse av romlig lokaliserte cAMP-signaler nesten utelukkende blitt utført i bare to romlige dimensjoner, og den aksiale cAMP-fordelingen har sjelden blitt vurdert som25. Dette skyldes sannsynligvis at lav SNR hindret muligheten til å måle og visualisere cAMP-graderinger i tre romlige dimensjoner. For å overvinne begrensninger ved bruk av FRET-sensorer med lav SNR, har vi implementert hyperspektral avbildning og analysetilnærminger for å måle FRET i enkeltceller25,26,27.
Hyperspektral avbildning tilnærminger ble utviklet av NASA for å skille terrestriske objekter til stede i satellittbilder28,29. Disse teknikkene har siden blitt oversatt til fluorescensmikroskopifeltet30, med flere kommersielle konfektmikroskopsystemer som tilbyr spektraldetektorer. I tradisjonell (ikke-spektral) fluorescensavbildning er prøven spent ved hjelp av et båndpassfilter eller en laserlinje, og utslippet samles ved hjelp av et annet båndpassfilter, ofte valgt for å matche topputslippsbølgelengden til fluorofor(e). Til sammenligning søker hyperspektrale avbildningsmetoder å prøve en komplett spektralprofil av enten fluorescensutslippet26,31,32 eller eksitasjon33,34 ved bestemte bølgelengdeintervaller. I våre tidligere studier viste vi at hyperspektral avbildning og analyse tilnærminger kan tilby forbedret kvantifisering av FRET-signaler i celler sammenlignet med tradisjonelle filterbaserte FRET-avbildningsteknikker26. Her presenterer vi en metodikk for å utføre 4-dimensjonale (x, y, z og λ) hyperspektral FRET-avbildning og analyse for å måle og visualisere cAMP-distribusjoner i tre romlige dimensjoner. Disse metodene har tillatt visualisering av agonistinduserte cAMP-romlige gradienter i enkeltceller25. Interessant, avhengig av agonisten, kan cAMP-gradienter være tydelige i celler. Metodikken som presenteres her benytter spektral unmixing av ikke-jevn bakgrunn og cellulær autofluorescence for å forbedre nøyaktigheten av FRET-målingene. Mens denne metodikken er demonstrert i lungemikrovaskulære endotelceller (PMVEC) ved hjelp av en cAMP FRET biosensor, kan metodikken enkelt endres for bruk med alternative FRET-reportere eller alternative cellelinjer.

<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

Syklisk AMP er en annen budbringer som er involvert i et bredt spekter av cellulære og fysiologiske aktiviteter. Flere studier tyder på at cAMP-signaler er inndelt, og at oppdeling bidrar til å signalisere spesifisitet innenfor cAMP-signalveien. Utviklingen av Fӧrster resonansenergioverføring (FRET) basert biosensorer har fremmet evnen til å måle og visualisere cAMP-signaler i celler. Disse målingene er imidlertid ofte begrenset til to romlige dimensjoner, noe som kan føre til feiltolkning av data. Til dags dato har det bare vært svært begrensede målinger av cAMP-signaler i tre romlige dimensjoner (x, y og z), på grunn av de tekniske begrensningene ved bruk av FRET-sensorer som iboende viser lavt signal-til-støy-forhold (SNR). I tillegg er tradisjonelle filterbaserte avbildningsmetoder ofte ineffektive for nøyaktig måling av cAMP-signaler i lokaliserte subcellulære områder på grunn av en rekke faktorer, inkludert spektral krysstale, begrenset signalstyrke og autofluorescens. For å overvinne disse begrensningene og la FRET-baserte biosensorer brukes med flere fluoroforer, har vi utviklet hyperspektrale FRET-avbildnings- og analysetilnærminger som gir spektral spesifisitet for beregning av FRET-effektivitet og evnen til å skille FRET-signaler fra forvirrende autofluorescens og/eller signaler fra ekstra fluorescerende etiketter. Her presenterer vi metodikken for implementering av hyperspektral FRET-avbildning samt behovet for å konstruere et passende spektralbibliotek som verken er undersamplet eller oversamplet for å utføre spektral unmixing. Mens vi presenterer denne metoden for måling av tredimensjonale cAMP-distribusjoner i pulmonale mikrovaskulære endotelceller (PMVEC), kan denne metoden brukes til å studere romlige fordelinger av cAMP i en rekke celletyper.

Denne protokollen beskriver bruken av hyperspektrale FRET-avbildnings- og analysemetoder for å måle cAMP-gradienter i tre romlige dimensjoner i levende celler. Det er flere viktige trinn som er involvert i å generere disse resultatene, som det kreves nøye oppmerksomhet for mens du analyserer og kvantifiserer dataene. Disse viktige trinnene inkluderer bygging av et passende spektralbibliotek, bakgrunnsspektral unmixing, terring for å identifisere cellekantlinjer og FRET-effektivitetsberegninger. ...

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 grunn av iboende lavt signal-til-støy-forhold (SNR) av Fӧrster resonansenergioverføringssensorer (FRET), har måling av cAMP-signaler vært utfordrende, spesielt i tre romlige dimensjoner. Her beskriver vi en hyperspektral FRET-avbildnings- og analysemetodikk som tillater måling av cAMP-distribusjon i tre romlige dimensjoner.

Måling av 3-dimensjonale cAMP-distribusjoner i levende celler ved hjelp av 4-dimensjonale (x, y, z og λ) Hyperspektral FRET-avbildning og -analyse

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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