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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Die Autoren danken Dr. Kees Jalink (The Netherlands Cancer Institute und van Leeuwenhoek Center for Advanced Microscopy, Amsterdam, Niederlande) für die Bereitstellung des H188 cAMP FRET Biosensors und Kenny Trinh (College of Engineering, University of South Alabama) für die technische Hilfe bei der Reduzierung der Zeit, die für die Ausführung unserer speziell entwickelten Programmierskripte erforderlich ist.
Die Autoren möchten die Finanzierungsquellen anerkennen: American Heart Association (16PRE27130004), National Science Foundation; (1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

Dieses Protokoll folgt Verfahren, die vom Institutional Animal Care and Use Committee der University of South Alabama genehmigt wurden.
1. Zell-, Proben- und Reagenzienvorbereitung für die Bildgebung
<ol><li>Isolieren Sie pulmonale mikrovaskuläre Endothelzellen (PMVECs) von Ratten wie zuvor beschrieben35. HINWEIS: Die Zellen wurden vom Zellkulturkern an der University of South Alabama, Mobile, AL, auf 100-mm-Zellkulturschalen isoliert und kultiviert.</li>
	<li>Sa...

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Die Entwicklung von FRET-Biosensoren hat die Messung und Visualisierung von zyklischen Nukleotidsignalen in einzelnen Zellen ermöglicht, und es gibt große Vielversprechende für die Visualisierung subzellulärer Signalereignisse13,22,37,38. Die Verwendung von FRET-Biosensoren birgt jedoch mehrere Einschränkungen, darunter die niedrigen Signal-Rausch-Eigenschaften vieler fluoreszierender prot...

Cyclisches Adenosinmonophosphat (cAMP) ist ein zweiter Botenstoff, der an wichtigen zellulären und physiologischen Prozessen wie Zellteilung, Kalziumzufluss, Gentranskription und Signaltransduktion beteiligt ist. Eine wachsende Zahl von Beweisen deutet auf die Existenz von cAMP-Kompartimenten in der Zelle hin, durch die die Signalspezifität erreicht wird1,2,3,4,5,6,7. Bis vor kurzem wurde die cAMP-Kompartimentierung auf der Grundlage verschiedener physiologischer oder zellulärer Effekte abgeleitet, die von verschiedenen G-gekoppelten Rezeptoragonisten induziert wurden8,9,10,11. In jüngerer Zeit haben FRET-basierte Fluoreszenzbildgebungssonden neue Ansätze für die direkte Messung und Beobachtung von cAMP-Signalen in einer Zelle12,13,14geliefert.
Förster-Resonanz-Energietransfer (FRET) ist ein physikalisches Phänomen, bei dem die Energieübertragung zwischen Donor- und Akzeptormolekülen in einer nicht-strahlenden Weise stattfindet, wenn sich die Moleküle in unmittelbarer Nähebefinden 15,16. Mit der Entwicklung von FRET-basierten Fluoreszenzindikatoren wurde dieses physikalische Phänomen in biologischen Anwendungen verwendet, um Protein-Protein-Interaktionen17, Protein-Co-Lokalisation18, Ca+2-Signalisierung 19, Genexpression20, Zellteilung21 und zyklische Nukleotidsignalisierung zu untersuchen. FRET-basierte cAMP-Indikatoren bestehen typischerweise aus einer cAMP-Bindungsdomäne, einem Donorfluorophor und einem Akzeptorfluorophor22. Zum Beispiel besteht der H188 cAMP-Sensor12,22, der in dieser Methodik verwendet wird, aus einer cAMP-Bindungsdomäne, die aus Epac gewonnen wird und zwischen Türkis- (Spender-) und Venus- (Akzeptor) Fluorophoren liegt. Bei basalen Bedingungen (ungebunden) sind Türkis und Venus in einer Ausrichtung, so dass FRET zwischen den Fluorophoren auftritt. Bei der Bindung von cAMP an die Bindungsdomäne tritt eine Konformationsänderung auf, so dass sich Türkis und Venus auseinander bewegen, was zu einer Abnahme der FRET führt.
FRET-basierte Bildgebungsansätze bieten ein vielversprechendes Werkzeug zur Untersuchung und Visualisierung von cAMP-Signalen innerhalb einer Zelle. Aktuelle FRET-basierte mikroskopische Bildgebungsverfahren sind jedoch oft nur teilweise erfolgreich, um eine ausreichende Signalstärke zu erreichen, um FRET mit subzellulärer räumlicher Klarheit zu messen. Dies ist auf mehrere Faktoren zurückzuführen, darunter die begrenzte Signalstärke vieler FRET-Reporter, die hohe Präzision, die erforderlich ist, um Änderungen der FRET-Effizienz genau zu quantifizieren, und das Vorhandensein von Störfaktoren wie zellulärer Autofluoreszenz23,24. Das Ergebnis ist oft ein FRET-Bild, das von schwachem SNR geplagt wird, was die Visualisierung subzellulärer Veränderungen in FRET sehr schwierig macht. Darüber hinaus wurde die Untersuchung von räumlich lokalisierten cAMP-Signalen fast ausschließlich in nur zwei räumlichen Dimensionen durchgeführt und die axiale cAMP-Verteilung wurde selten berücksichtigt25. Dies liegt wahrscheinlich daran, dass ein niedriger SNR die Fähigkeit behinderte, cAMP-Gradienten in drei räumlichen Dimensionen zu messen und zu visualisieren. Um die Einschränkungen bei der Verwendung von FRET-Sensoren mit niedrigem SNR zu überwinden, haben wir hyperspektrale Bildgebungs- und Analyseansätze zur Messung von FRET in einzelnen Zellen25,26,27implementiert.
Hyperspektrale Bildgebungsansätze wurden von der NASA entwickelt, um terrestrische Objekte in Satellitenbildern zu unterscheiden28,29. Diese Techniken wurden seitdem in das Fluoreszenzmikroskopiefeld30übersetzt, wobei mehrere kommerzielle konfokale Mikroskopsysteme Spektraldetektoren anbieten. Bei der traditionellen (nicht-spektralen) Fluoreszenzbildgebung wird die Probe mit einem Bandpassfilter oder einer Laserlinie angeregt, und die Emission wird mit einem zweiten Bandpassfilter gesammelt, der oft so ausgewählt wird, dass er der Maximalen Emissionswellenlänge des Fluorophors entspricht. Im Gegensatz dazu versuchen hyperspektrale Bildgebungsansätze, ein vollständiges spektrales Profil entweder der Fluoreszenzemission26,31,32 oder der Anregung 33,34in bestimmten Wellenlängenintervallen zu testen. In unseren früheren Studien haben wir gezeigt, dass hyperspektrale Bildgebungs- und Analyseansätze im Vergleich zu herkömmlichen filterbasierten FRET-Bildgebungsverfahren eine verbesserte Quantifizierung von FRET-Signalen in Zellen bieten können26. Hier stellen wir eine Methodik zur Durchführung von 4-dimensionalen (x, y, z und λ) hyperspektralen FRET-Bildgebung und -Analyse vor, um cAMP-Verteilungen in drei räumlichen Dimensionen zu messen und zu visualisieren. Diese Ansätze haben die Visualisierung von agonisteninduzierten cAMP-Raumgradienten in einzelnen Zellenermöglicht 25. Interessanterweise können je nach Agonist cAMP-Gradienten in Zellen sichtbar sein. Die hier vorgestellte Methodik nutzt die spektrale Entmischung von ungleichmäßigem Hintergrund und zellulärer Autofluoreszenz, um die Genauigkeit der FRET-Messungen zu verbessern. Während diese Methodik in pulmonalen mikrovaskulären Endothelzellen (PMVECs) mit einem cAMP FRET-Biosensor demonstriert wird, könnte die Methodik leicht für die Verwendung mit alternativen FRET-Reportern oder alternativen Zelllinien modifiziert werden.

<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 ist ein zweiter Botenstoff, der an einer Vielzahl von zellulären und physiologischen Aktivitäten beteiligt ist. Mehrere Studien deuten darauf hin, dass cAMP-Signale kompartimentiert sind und dass die Kompartimentierung zur Signalspezifität innerhalb des cAMP-Signalwegs beiträgt. Die Entwicklung von FRET-basierten Biosensoren (Fӧrster resonance energy transfer) hat die Fähigkeit zur Messung und Visualisierung von cAMP-Signalen in Zellen gefördert. Diese Messungen beschränken sich jedoch oft auf zwei räumliche Dimensionen, was zu Fehlinterpretationen von Daten führen kann. Bisher gab es nur sehr begrenzte Messungen von cAMP-Signalen in drei räumlichen Dimensionen (x, y und z), aufgrund der technischen Einschränkungen bei der Verwendung von FRET-Sensoren, die von Natur aus ein niedriges Signal-Rausch-Verhältnis (SNR) aufweisen. Darüber hinaus sind herkömmliche filterbasierte Bildgebungsansätze aufgrund einer Reihe von Faktoren, einschließlich spektralem Übersprechen, begrenzter Signalstärke und Autofluoreszenz, oft unwirksam für die genaue Messung von cAMP-Signalsignalen in lokalisierten subzellulären Regionen. Um diese Einschränkungen zu überwinden und die Verwendung von FRET-basierten Biosensoren mit mehreren Fluorophoren zu ermöglichen, haben wir hyperspektrale FRET-Bildgebungs- und Analyseansätze entwickelt, die spektrale Spezifität für die Berechnung von FRET-Wirkungsgraden und die Fähigkeit bieten, FRET-Signale spektral von verwirrender Autofluoreszenz und / oder Signalen von zusätzlichen fluoreszierenden Markierungen zu trennen. Hier stellen wir die Methodik zur Implementierung der hyperspektralen FRET-Bildgebung sowie die Notwendigkeit vor, eine geeignete Spektralbibliothek zu konstruieren, die weder unter- noch übersampelt ist, um eine spektrale Entmischung durchzuführen. Während wir diese Methodik zur Messung dreidimensionaler cAMP-Verteilungen in pulmonalen mikrovaskulären Endothelzellen (PMVECs) vorstellen, könnte diese Methodik verwendet werden, um räumliche Verteilungen von cAMP in einer Reihe von Zelltypen zu untersuchen.

Dieses Protokoll beschreibt die Verwendung hyperspektraler FRET-Bildgebungs- und Analyseansätze zur Messung von cAMP-Gradienten in drei räumlichen Dimensionen in lebenden Zellen. Bei der Generierung dieser Ergebnisse gibt es mehrere wichtige Schritte, für die bei der Analyse und Quantifizierung der Daten sorgfältige Aufmerksamkeit erforderlich ist. Zu diesen Schlüsselschritten gehören der Aufbau einer geeigneten Spektralbibliothek, die spektrale Entmischung im Hintergrund, die Schwellenwerte zur Identifizierung von...

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

Aufgrund des inhärenten niedrigen Signal-Rausch-Verhältnisses (SNR) von Fӧrster-Resonanz-Energieübertragungssensoren (FRET) war die Messung von cAMP-Signalen eine Herausforderung, insbesondere in drei räumlichen Dimensionen. Hier beschreiben wir eine hyperspektrale FRET-Bildgebungs- und Analysemethodik, die die Messung der cAMP-Verteilung in drei räumlichen Dimensionen ermöglicht.

Messung von 3-dimensionalen cAMP-Verteilungen in lebenden Zellen mittels 4-dimensionaler (x, y, z und λ) hyperspektraler FRET-Bildgebung und -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.

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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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