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

Gli autori desiderano ringraziare il Dr. Kees Jalink (The Netherlands Cancer Institute e van Leeuwenhoek Center for Advanced Microscopy, Amsterdam, Paesi Bassi) per averci fornito il biosensore H188 cAMP FRET e Kenny Trinh (College of Engineering, University of South Alabama) per l'aiuto tecnico nel ridurre il tempo impiegato per eseguire i nostri script di programmazione sviluppati su misura.
Gli autori desiderano riconoscere le fonti di finanziamento: American Heart Association (16PRE27130004), National Science Foundation; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

Questo protocollo segue le procedure approvate dal Comitato istituzionale per la cura e l'uso degli animali della University of South Alabama.
1. Preparazione di cellule, campioni e reagenti per l'imaging
<ol><li>Isolare le cellule endoteliali microvascolari polmonari di ratto (PMVEC) come descritto in precedenza35. NOTA: Le cellule sono state isolate e coltivate dal Cell Culture Core presso la University of South Alabama, Mobile, AL su piatti di coltura cellulare ...

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Lo sviluppo di biosensori FRET ha permesso la misurazione e la visualizzazione di segnali nucleotidici ciclici in singole cellule, e c'è una grande promessa per la visualizzazione degli eventi di segnalazione subcellulare13,22,37,38. Tuttavia, l'uso di biosensori FRET presenta diverse limitazioni, tra cui le basse caratteristiche segnale-rumore di molti reporter FRET fluorescenti basati su pro...

L'adenosina monofosfato ciclico (cAMP) è un secondo messaggero coinvolto nei principali processi cellulari e fisiologici tra cui la divisione cellulare, l'afflusso di calcio, la trascrizione genica e la trasduzione del segnale. Un numero crescente di prove suggerisce l'esistenza di compartimenti cAMP nella cellula attraverso i quali si raggiunge la specificità di segnalazione1,2,3,4,5,6,7. Fino a poco tempo fa, la compartimentazione cAMP era dedotta sulla base di distinti effetti fisiologici o cellulari indotti da diversi agonisti del recettore accoppiato G8,9,10,11. Più recentemente, le sonde di imaging a fluorescenza basate su FRET hanno fornito nuovi approcci per la misurazione diretta e l'osservazione dei segnali cAMP in una cella12,13,14.
Il trasferimento di energia di risonanza di Förster (FRET) è un fenomeno fisico in cui il trasferimento di energia avviene tra molecole donatrici e accettori in modo non radiativo quando le molecole sono in prossimità15,16. Con lo sviluppo di indicatori fluorescenti basati su FRET, questo fenomeno fisico è stato utilizzato in applicazioni biologiche per studiare le interazioni proteina-proteina17, co-localizzazione proteica18, Ca+2 segnalazione19, espressionegenica 20, divisione cellulare21 e segnalazione nucleotidica ciclica. Gli indicatori cAMP basati su FRET sono tipicamente costituiti da un dominio di legame cAMP, un fluoroforo donatore e un fluoroforo accettore22. Ad esempio, il sensore H188 cAMP12,22 utilizzato in questa metodologia è costituito da un dominio di legame cAMP ottenuto da Epac, inserito tra fluorofori Turchese (donatore) e Venere (accettore). In condizioni basali (non legate), Turchese e Venere sono in un orientamento tale che FRET si verifica tra i fluorofori. Al momento del legame di cAMP al dominio di legame, si verifica un cambiamento conformazionale tale che Turchese e Venere si allontanano con conseguente diminuzione di FRET.
Gli approcci di imaging basati su FRET offrono uno strumento promettente per indagare e visualizzare i segnali cAMP all'interno di una cellula. Tuttavia, le attuali tecniche di imaging microscopico basate su FRET hanno spesso solo parzialmente successo nel raggiungere una potenza del segnale sufficiente per misurare FRET con chiarezza spaziale subcellulare. Ciò è dovuto a diversi fattori, tra cui la limitata potenza del segnale di molti reporter FRET, l'alto livello di precisione richiesto per quantificare con precisione i cambiamenti nell'efficienza FRET e la presenza di fattori confondenti, come l'autofluorescenza cellulare23,24. Il risultato è spesso un'immagine FRET che è afflitta da SNR debole, rendendo molto difficile la visualizzazione dei cambiamenti subcellulari in FRET. Inoltre, lo studio dei segnali cAMP localizzati spazialmente è stato eseguito quasi esclusivamente in due sole dimensioni spaziali e la distribuzione assiale cAMP è stata raramenteconsiderata 25. Ciò è probabilmente dovuto al fatto che un basso SNR ha impedito la capacità di misurare e visualizzare i gradienti cAMP in tre dimensioni spaziali. Per superare i limiti dell'utilizzo di sensori FRET con basso SNR, abbiamo implementato approcci di imaging e analisi iperspettrali per misurare FRET in singole celle25,26,27.
Gli approcci di imaging iperspettrale sono stati sviluppati dalla NASA per differenziare gli oggetti terrestri presenti nelle immaginisatellitari28,29. Da allora queste tecniche sono state tradotte nel campo di microscopia a fluorescenza30, con diversi sistemi di microscopio confocale commerciali che offrono rivelatori spettrali. Nell'imaging a fluorescenza tradizionale (non spettrale), il campione viene eccitato utilizzando un filtro passa-banda o una linea laser e l'emissione viene raccolta utilizzando un secondo filtro passa-banda, spesso selezionato per corrispondere alla lunghezza d'onda di emissione di picco del fluoroforo (s). Al contrario, gli approcci di imaging iperspettrale cercano di campionare un profilo spettrale completo dell'emissione di fluorescenza26,31,32 o dell'eccitazione33,34 a specifici intervalli di lunghezza d'onda. Nei nostri studi precedenti, abbiamo dimostrato che gli approcci di imaging e analisi iperspettrali possono offrire una migliore quantificazione dei segnali FRET nelle cellule rispetto alle tradizionali tecniche di imaging FRET basate su filtro26. Qui presentiamo una metodologia per l'esecuzione di imaging e analisi FRET iperspettrali 4-dimensionali (x, y, z e λ) per misurare e visualizzare le distribuzioni cAMP in tre dimensioni spaziali. Questi approcci hanno permesso la visualizzazione di gradienti spaziali cAMP indotti da agonisti in singole cellule25. È interessante notare che, a seconda dell'agonista, i gradienti cAMP possono essere evidenti nelle cellule. La metodologia qui presentata utilizza l'unmixing spettrale di fondo non uniforme e l'autofluorescenza cellulare per migliorare l'accuratezza delle misurazioni FRET. Mentre questa metodologia è dimostrata nelle cellule endoteliali microvascolari polmonari (PMVEC) utilizzando un biosensore cAMP FRET, la metodologia potrebbe essere facilmente modificata per l'uso con reporter FRET alternativi o linee cellulari alternative.

<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

L'AMP ciclico è un secondo messaggero che è coinvolto in una vasta gamma di attività cellulari e fisiologiche. Diversi studi suggeriscono che i segnali cAMP sono compartimentati e che la compartimentazione contribuisce alla specificità della segnalazione all'interno della via di segnalazione cAMP. Lo sviluppo di biosensori basati sul trasferimento di energia di risonanza Fӧrster (FRET) ha favorito la capacità di misurare e visualizzare i segnali cAMP nelle cellule. Tuttavia, queste misurazioni sono spesso limitate a due dimensioni spaziali, il che può comportare un'interpretazione errata dei dati. Ad oggi, ci sono state solo misurazioni molto limitate dei segnali cAMP in tre dimensioni spaziali (x, y e z), a causa delle limitazioni tecniche nell'utilizzo dei sensori FRET che mostrano intrinsecamente un basso rapporto segnale/rumore (SNR). Inoltre, i tradizionali approcci di imaging basati su filtri sono spesso inefficaci per la misurazione accurata dei segnali cAMP nelle regioni subcellulari localizzate a causa di una serie di fattori, tra cui la diafonia spettrale, la potenza limitata del segnale e l'autofluorescenza. Per superare queste limitazioni e consentire l'utilizzo di biosensori basati su FRET con più fluorofori, abbiamo sviluppato approcci di imaging e analisi FRET iperspettrali che forniscono specificità spettrale per il calcolo delle efficienze FRET e la capacità di separare spettralmente i segnali FRET dall'autofluorescenza confondente e / o segnali da etichette fluorescenti aggiuntive. Qui, presentiamo la metodologia per l'implementazione dell'imaging FRET iperspettrale e la necessità di costruire una libreria spettrale appropriata che non sia né sottocampionata né sovracampionata per eseguire l'unmixing spettrale. Mentre presentiamo questa metodologia per la misurazione delle distribuzioni tridimensionali di cAMP nelle cellule endoteliali microvascolari polmonari (PMVEC), questa metodologia potrebbe essere utilizzata per studiare le distribuzioni spaziali di cAMP in una gamma di tipi di cellule.

Questo protocollo descrive l'uso di approcci iperspettrali di imaging e analisi FRET per misurare i gradienti cAMP in tre dimensioni spaziali nelle cellule viventi. Ci sono diversi passaggi chiave coinvolti nella generazione di questi risultati, per i quali è necessaria un'attenta attenzione durante l'analisi e la quantificazione dei dati. Questi passaggi chiave includono la costruzione di una libreria spettrale appropriata, lo smixing spettrale di sfondo, la soglia per identificare i bordi delle celle e i calcoli dell'...

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

A causa del basso rapporto segnale-rumore (SNR) dei sensori basati sul trasferimento di energia di risonanza Fӧrster (FRET), la misurazione dei segnali cAMP è stata impegnativa, specialmente in tre dimensioni spaziali. Qui, descriviamo una metodologia di imaging e analisi FRET iperspettrale che consente la misurazione della distribuzione cAMP in tre dimensioni spaziali.

Misurazione di distribuzioni cAMP tridimensionali in cellule viventi utilizzando imaging e analisi FRET iperspettrale a 4 dimensioni (x, y, z e λ)

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.

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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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