Name: measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis
Uploaded: 2020-10-27T13:30:00
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 gerne anerkende Dr. Kees Jalink (The Netherlands Cancer Institute og van Leeuwenhoek Center for Advanced Microscopy, Amsterdam, Holland) for at give os H188 cAMP FRET biosensor og Kenny Trinh (College of Engineering, University of South Alabama) for teknisk hjælp til at reducere den tid, det tog at køre vores specialudviklede programmering scripts.
Forfatterne vil gerne anerkende finansieringskilderne: American Heart Association (16PRE27130004), National Science Foundation; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

Denne protokol følger procedurer godkendt af University of South Alabama Institutional Animal Care and Use Committee.
1. Fremstilling af celler, prøver og reagenser til billeddannelse
<ol><li>Isoler rottepulmonale mikrovaskulære endotelceller (PMVECs) som beskrevet tidligere35. BEMÆRK: Celler blev isoleret og kultiveret af Cell Culture Core ved University of South Alabama, Mobile, AL på 100 mm cellekultur retter.</li>
	<li>Frø isolerede PMVECs på 25 mm runde...

<ol>
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Udviklingen af FRET biosensorer har gjort det muligt at måle og visualisere cykliske nukleotidsignaler i enkelte celler, og der er store løfter om at visualisere subcellulære signalhændelser13,22,37,38. Brugen af FRET biosensorer præsenterer imidlertid flere begrænsninger, herunder de lave signal-til-støj-egenskaber hos mange fluorescerende proteinbaserede FRET-reportere og FRET-journali...

Cyklisk adenosin monophosphat (cAMP) er en anden messenger involveret i centrale cellulære og fysiologiske processer, herunder celledeling, calcium tilstrømning, gen transskription, og signal transduktion. En voksende mængde af beviser tyder på eksistensen af cAMP rum icellen,hvorigennem signalering specificitet opnås1,2,3,4,5,6,7. Indtil for nylig blev cAMP-opdeling udledt baseret på forskellige fysiologiske eller cellulære virkninger induceret af forskellige G-koblede receptor-agonister8,9,10,11. For nylig har FRET-baserede fluorescensbilledsonder givet nye tilgange til direkte måling og observation af cAMP-signaler i en celle12,13,14.
Förster resonans energioverførsel (FRET) er et fysisk fænomen, hvor energioverførsel sker mellem donor og acceptor molekyler i en ikke-radiativ måde, når molekylerne er i nærheden15,16. Med udviklingen af FRET-baserede fluorescerende indikatorer er dette fysiske fænomen blevet brugt i biologiske anvendelser til at studere protein-protein interaktioner17,protein co-lokalisering18,Ca+2 signalering19, genekspression20, celledeling21 og cyklisk nukleotid signalering. FRET-baserede cAMP-indikatorer består typisk af et cAMP-bindingsdomæne, en donorfluohore og en acceptorfluoriphe22. For eksempel består H188 cAMP sensor12,22, der anvendes i denne metode, af et cAMP-bindingsdomæne fremstillet af Epac, klemt inde mellem turkis (donor) og Venus (acceptor) fluorophorer. Ved basale forhold (ubundet) er turkis og Venus i en retning, så FRET opstår mellem fluorophorerne. Ved binding af cAMP til bindingsdomænet sker der en kropsbygningsændring, så Turkis og Venus bevæger sig fra hinanden, hvilket resulterer i et fald i FRET.
FRET-baserede billedbehandlingsmetoder er et lovende værktøj til at undersøge og visualisere cAMP-signaler i en celle. Men, nuværende FRET baseret mikroskopiske billeddannelse teknikker er ofte kun delvis vellykket i at opnå tilstrækkelig signalstyrke til at måle FRET med subcellulære rumlige klarhed. Dette skyldes flere faktorer, herunder den begrænsede signalstyrke hos mange FRET-journalister, det høje præcisionsniveau, der kræves for præcist at kvantificere ændringer i FRET-effektivitet, og tilstedeværelsen af forvirrende faktorer, såsom cellulær autofluorescence23,24. Resultatet er ofte et FRET-billede, der er plaget af svag SNR, hvilket gør visualisering af subcellulære ændringer i FRET meget vanskelige. Desuden er undersøgelsen af rumligt lokaliserede cAMP-signaler næsten udelukkende blevet udført i to rumlige dimensioner, og den aksiale cAMP-fordeling er sjældent blevet betragtet som25. Dette skyldes sandsynligvis, at lav SNR hindrede evnen til at måle og visualisere cAMP-gradienter i tre rumlige dimensioner. For at overvinde begrænsninger ved at bruge FRET-sensorer med lav SNR har vi implementeret hyperspektral billeddannelse og analysemetoder til måling af FRET i enkeltceller25,26,27.
Hyperspektral billeddannelse tilgange blev udviklet af NASA til at differentiere jordbaserede objekter til stede i satellitbilleder28,29. Disse teknikker er siden blevet oversat til fluorescensmikroskopifelt30, med flere kommercielle konfokale mikroskopsystemer, der tilbyder spektraldetektorer. I traditionelle (ikke-spektrale) fluorescens imaging, prøven er ophidset ved hjælp af et band-pass filter eller en laser linje, og emissionen indsamles ved hjælp af en anden band-pass filter, ofte udvalgt til at matche peak emission bølgelængde af fluorophore (r). I modsætning hertil søger hyperspektral billeddannelsesmetoder at prøve en komplet spektral profil af enten fluorescensemissionen26,31,32 eller excitation33,34 med bestemte bølgelængdeintervaller. I vores tidligere undersøgelser viste vi, at hyperspektral billeddannelse og analyse tilgange kan tilbyde forbedret kvantificering af FRET-signaler i celler i forhold til traditionelle filter-baserede FRET billeddannelse teknikker26. Her præsenterer vi en metode til udførelse af 4-dimensionelle (x, y, z og λ) hyperspektral FRET billeddannelse og analyse til at måle og visualisere cAMP fordelinger i tre rumlige dimensioner. Disse tilgange har gjort det muligt visualisering af agonistiske-inducerede cAMP rumlige gradienter i enkeltceller25. Interessant, afhængigt af agonist, cAMP gradienter kan være synlige i celler. Den metode, der præsenteres her, udnytter spektral blanding af ikke-ensartet baggrund og cellulær autofluorescence til at forbedre nøjagtigheden af FRET-målingerne. Mens denne metode er demonstreret i lungemikrovaskulære endotelceller (PMVECs) ved hjælp af en cAMP FRET biosensor, kan metoden let ændres til brug med alternative FRET-journalister 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

Cyklisk AMP er en anden messenger, der er involveret i en bred vifte af cellulære og fysiologiske aktiviteter. Flere undersøgelser tyder på, at cAMP-signaler er opdelt, og at opdeling bidrager til at signalere specificitet inden for cAMP-signalvejen. Udviklingen af Fӧrster resonans energioverførsel (FRET) baseret biosensorer har fremmet evnen til at måle og visualisere cAMP signaler i celler. Disse målinger er dog ofte begrænset til to rumlige dimensioner, hvilket kan resultere i fejlfortolkning af data. Til dato har der kun været meget begrænsede målinger af cAMP-signaler i tre rumlige dimensioner (x, y og z) på grund af de tekniske begrænsninger ved at bruge FRET-sensorer, der i sagens natur udviser lavt signal til støjforhold (SNR). Derudover er traditionelle filterbaserede billeddannelsesmetoder ofte ineffektive til nøjagtig måling af cAMP-signaler i lokaliserede subcellulære områder på grund af en række faktorer, herunder spektral krydstale, begrænset signalstyrke og autofluorescence. For at overvinde disse begrænsninger og tillade FRET-baserede biosensorer, der skal anvendes med flere fluorophorer, har vi udviklet hyperspektrale FRET billed- og analysemetoder, der giver spektral specificitet til beregning af FRET-effektivitet og evnen til at spektralt adskille FRET-signaler fra forvirrende autofluorescence og / eller signaler fra yderligere fluorescerende etiketter. Her præsenterer vi metoden til implementering af hyperspektral FRET-billeddannelse samt behovet for at konstruere et passende spektralbibliotek, der hverken er undersampled eller oversampled for at udføre spektral ublandet. Mens vi præsenterer denne metode til måling af tredimensionelle cAMP-fordelinger i lungemikrovaskulære endotelceller (PMVECs), kan denne metode bruges til at studere rumlige fordelinger af cAMP i en række celletyper.

Denne protokol beskriver brugen af hyperspektrale FRET-billed- og analysemetoder til måling af cAMP-gradienter i tre rumlige dimensioner i levende celler. Der er flere vigtige trin involveret i at generere disse resultater, for hvilke der kræves omhyggelig opmærksomhed, mens du analyserer og kvantificerer dataene. Disse vigtige trin omfatter opførelse af et passende spektralbibliotek, baggrundsspektral spektral ub blanding, tærskeling for at identificere cellekanter og FRET-effektivitetsberegninger. <strong class="x...

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 af iboende lave signal-til-støj-forhold (SNR) af Fӧrster resonans energioverførsel (FRET) baserede sensorer, måling af cAMP signaler har været udfordrende, især i tre rumlige dimensioner. Her beskriver vi en hyperspektral FRET billed- og analysemetode, der gør det muligt at måle cAMP-distribution i tre rumlige dimensioner.

Måling af 3-dimensionelle cAMP-fordelinger i levende celler ved hjælp af 4-dimensionelle (x, y, z og λ) Hyperspektral FRET Imaging and Analysis

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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