We thank Dr. Gilles Dupuis for critical reading of the manuscript. 
Funding
This work was supported by the Canadian Institutes for Health Research, grant # MOP-126173. C.M.D is a member of the Fonds de la Recherche en Sant&#233; (FRSQ)-funded Centre de Recherche Clinique &#201;tienne LeBel.

1. Herstellung von Lösungen für Cellular pH-Kalibrierung <ol><li> Fügen Sie die folgenden Verbindungen zu fünf separate 50 ml konische Röhrchen bis 5 Lösungen für die Kalibrierung verwendet werden vorbereitet: <ul><li> NaCl (1 ml von 1 M NaCl) (1 M NaCl = 0,58 g in 10 ml H 2 0) </li><li> KCl (6,75 ml 1 M KCl) (1 M KCl = 0,75 g in 10 ml H 2 0) </li><li> Glucose (1 ml von 1 M D-Glucose) (1 M D-Glucose = 1,80 g in 10 ml H 2 0) </li><li> MgS0 4 (0,05 ml von 1 M MgSO 4) (1 M MgSO 4 = 1,20 g in 10 ml H 2 0) </li><li> 20 mM HEPES (1 ml von 1 M HEPES) (1 M HEPES = 2,38 g in 10 ml H 2 0) </li><li> CaCl 2 (0,05 ml 1 M CaCl 2) (1 M CaCl 2 = 1,47 g in 10 ml H 2 0) Bereiten alle Lösungen in doppelt destilliertem H 2 O (ddH 2 O). </li></ul></li><li> Füllen Sie jede Lösung mit 35 ml ddH2O und mit einem Magnetrührer mischen, bis soLösung erreicht wird. </li><li> Der pH-Wert jeder Lösung mit 1 N KOH oder 1 N HCl auf pH-Werte von 5,5, 6,0, 6,5, 7,0 und 7,5 erhalten. Dann wird das Volumen mit destilliertem Wasser auf ein Endvolumen von 50 ml einzustellen. </li><li> Maßnahme 5 mg Nigericin und es auf 670 ul absolutem EtOH hinzufügen, um eine 10 mM Stammlösung Nigericin vorzubereiten. Achtung: Nigericin ist hochtoxisch und müssen mit großer Sorgfalt (Handschuhe, Schutzbrille und Maske) behandelt werden. Mische die Lösung durch Umdrehen bis die Nigericin vollständig gelöst ist. </li><li> In 0,05 ml 10 mM Nigericin (Endkonzentration 10 uM), um die Eichlösungen. Ionophor Nigericin ist, die Permeabilität der Zellmembran erhöht und bewirkt Äquilibrierung von intrazellulären und extrazellulären pH-Wert in Gegenwart eines depolarisierenden Konzentration der extrazellulären K +. </li><li> Eichlösungen kann bis zu 1 Monat bei 4 ° C gelagert werden, </li></ol> 2. Zellpräparation <ol><li> Unter einer biologischen safety Haube, Samenzellen in 35 mm x 10 mm Kulturschalen in 2 ml Wachstumsmedium mit 10% fötalem Rinderserum und Antibiotika ergänzt. Verwenden der Menge von Zellen benötigt wird, um ungefähr 50% Konfluenz nach 24 Stunden zu gewährleisten. HINWEIS: In unseren Experimenten verwenden wir die HT-1080-Zelllinie und Platte 1 x 10 5 Zellen pro Kulturschale. </li><li> Bestimmen der Anzahl von Kulturschalen für das Experiment und die Platte 5 zusätzliche Kulturschalen für die Kalibrierung verwendet werden, benötigt. </li><li> Ort Kulturschalen bei 37 ° C / 5% CO 2-Inkubator für mindestens 24 Stunden. </li></ol> 3. Herstellung von pH-Messsonden <ol><li> Bereiten Sie eine 1 M Stammlösung des fluoreszierenden pH-Sonde 8-Hydroxypyren-1, 3,6 trisulfonsäure, Trinatriumsalzes (HPTS / Pyranin) in ddH 2 0. Diese Lösung muss bei RT gelagert und vor Licht geschützt werden. </li><li> Bereiten Sie eine Stammlösung von 1 mM 5 - (und-6)-Carboxy seminaphthorhodafluor Acetoxymethylester Acetat (C-SNARF-01.00; genannt Thereâach SNARF-1) durch Lösen von 1 mg SNARF-1 in 1,76 ml DMSO. Durch wiederholtes Pipettieren gründlich mischen. SNARF-1-Lösung muss bei -20 ° C gelagert und vor Licht geschützt werden. </li></ol> 4. Zellmarkierung <ol><li> Sechzehn Stunden vor der Live-Cell-Imaging, fügen 2 ul 1M HPTS Stammlösung (Endkonzentration 1 mM) zu jeder Kulturschale mit 2 ml Vollmedium. </li><li> Bringen Sie die Kulturschalen in den Inkubator. Die HPTS Sonde zell impermeante, daher Label Organellen muss die Sonde durch pinocytosis genommen werden. Diese Bedingung erfordert, dass die Zell Inkubationen mit HPTS in serumhaltigem Kulturmedium durchgeführt. </li><li> Nach der Inkubation mit HPTS, waschen Sie die Zellen zweimal mit sterilem PBS und 2 ml Serum-freiem Medium für mindestens 2 Stunden. Inkubation mit SNARF-1 benötigt, um in serumfreiem Medium durchgeführt, da Serum kann die Aufnahme der Sonde zu verhindern. </li><li> In 10 ul einer 1 mM SNARF-1-Stammlösung zu erhalten afinal Konzentration von 5 uM und lassen Zellen für 20 min bei 37 ° C unter schwachem Licht inkubiert. C-SNARF-1-Konzentration und Inkubationszeit kann je nach Zelltyp variieren. </li><li> Am Ende der Inkubation wurden die Zellen zweimal Waschen in sterilem PBS und 2 ml serumfreies Medium. </li></ol> 5. Zugabe von pH-Modulatoren <ol><li> Um zu beurteilen, ob die Methode können pH-Veränderungen in den Zellen erkennen können verschiedene Reagenzien, die intrazellulären pH-Modulation verwendet werden: <ul><li> Bafilomycin A1, ein spezifischer Inhibitor der V-ATPase-5 </li><li> Na + / H +-Austauscher Isoform 1 (NHE-1) 6 </li><li> NH 4 Cl, eine schwache Base, die intrazelluläre pH 7 erhöht </li></ul><ol><li> Stocklösungen der oben genannten Reagenzien: <ul><li> Bafilomycin A1: 160 ul DMSO hinzufügen 10 ug Bafilomycin A1 um eine Stammlösung von 100 uM erhalten </li><li> EIPA: add 25 mg EIPA auf 1,67 ml Methanol obtaina Endkonzentration von 50 mM </li><li> NH 4 Cl: Man löst 0,53 g Ammoniumchlorid in 10 ml ddH 2 O auf eine Endkonzentration von 1 mM zu erhalten. </li></ul> Hinweis: Alle Stammlösungen sind bei 4 ° C aufbewahrt werden, außer Bafilomycin A1, die bei -20 ° C gelagert und vor Licht geschützt werden müssen. </li><li> In getrennten Kulturschalen, fügen Sie eines der Reagenzien, wie unten beschrieben: <ul><li> 2 ul 100 uM Bafilomycin A1 (Endkonzentration 100 nM) </li><li> 1 ul 50 mM EIPA (Endkonzentration 25 uM) </li><li> 40 ul 1 M NH 4 Cl-Lösung (Endkonzentration 20 mM) </li></ul></li><li> Die Zellen für 30 min bei 37 ° C. </li></ol></li><li> Fahren Sie mit der Bildaufnahme. </li></ol> 6. Technische Daten des Live-Cell-Imaging-Mikroskop und Akquisition Einstellungen <ol><li> Führen Sie Live-Cell-Imaging mit ein 40X-Objektiv auf einer spektralen invertierten konfokalen Mikroskopie montiertpe mit einem motorisierten XY-Tisch, eine Kammer, die mit Glasboden Kulturschalen in einer kontrollierten Umweltbedingungen (Temperatur, Luftfeuchtigkeit, CO 2)-Stufe Inkubator bietet Platz ausgestattet. </li><li> Das konfokale Instrument (Olympus FV1000) verwendet, hier ist ausgestattet mit: <ul><li> Ein Diodenlaser 405 nm </li><li> A 40 mW Argon-Laser (458 nm, 488 nm, 515 nm) </li><li> Ein Helium-Neon-Laser (543 nm) </li><li> Fluorit-10X-Objektiv, NA0.4 </li><li> Ein LUCPLFLN 40X-Objektiv, NA0.60 </li><li> Ein PLAPONSC PLAN APO 60x Öl-Objektiv, NA1.4 </li><li> Ein dichroitischen Spiegel DM 405/458/488/543 </li><li> Ein SDM 560 und ein SDM 640 dichroitischen Filter </li><li> Ein BA655-755-Sperrfilter </li><li> Ein Strahlteiler BS20/80 </li><li> Datenanalyse-Software für die Bildaufnahme und-analyse, </li></ul></li><li> Sobald die Bühne Inkubator hat bis zu 37 ° C erwärmt und das Mikroskop aufgebaut ist, legen Sie eine Kulturschale zuvor mit HPTS und SNARF-1 in der konfokalen Imaging-Bühne Kammer inkubiert. </li><li> Aach Ortung einen Bereich von Interesse, stellen die Anschaffungs Einstellungen Software Abbildung 1. <ul><li> Richten Sie zwei virtuelle Kanalsätze. </li><li> Initiieren Sie die erste Phase, die HPTS Anregung bei 405 nm und Emission bei 505 entspricht Sammlung - 525 nm und SNARF-1-Anregung bei 543 nm und Emissions Kollektion bei 570 bis 600 nm auf. </li><li> Initiieren Sie die zweite Phase, die HPTS Anregung bei 458 nm und Emission bei 505 entspricht Sammlung - 525 nm und SNARF-1-Anregung bei 543 nm und Emissions Kollektion bei 640 bis 650 nm auf. </li><li> Das konfokale Blende ist standardmäßig auf 175 um eingestellt und optimale Blende muss auf 300 um manuell eingestellt werden. </li><li> Nehmen Bild von Stapelserien horizontale optische Schnitte (800 x 800 Pixel) von 0,4 um Dicke. </li></ul></li></ol> HINWEIS: Ein 40X-Objektiv ist sinnvoll, mehrere Zellen gleichzeitig zu beobachten und funktioniert gut für Organelle Betrachtung. , Räumliche Auflösung und Signalintensität sind jedoch better erreicht mit einem 60x Öl-Objektiv. Darüber hinaus gibt es keine Anregung oder Emission Übersprechen zwischen den beiden Farbstoffen. 7. Kalibrierung von pH-Messsonden <ol><li> Vor der Kalibrierung entfernen Medium von der Kulturschale zuvor mit HPTS und SNARF-1 inkubiert, zweimal mit sterilem PBS und dann 2 ml einer der fünf Kalibrierungslösungen. </li><li> Legen Sie die Kulturschale in die konfokale Stufenkammer. </li><li> Fahren Sie mit Zeitraffer-Bildaufnahme nach den folgenden Einstellungen: <ol><li> Stellen Sie die Anzahl der Bilder und Intervalldauer Zeiten für das Experiment. Hinweis: Wir verwenden in der Regel eine 1 min Intervall zwischen den Bildern für bis zu 30 min. </li><li> Programmieren Sie den Auslöser, um zwischen dem Erwerb von jedem Bild zu schließen. </li></ol></li><li> Notieren Sie sich die Fluoreszenzintensität pro min in einem Tabellenkalkulationssoftware und bestimmen Sie die Zeit, in der Fluoreszenzintensität ist stabil. Die intrazelluläre pH-Wert in der Regel nach 15 stabil - 20 min. </li><li> REPEAT Schritte 7.1 bis 7.4 mit einer neuen Kulturschale für jede verbleibende Kalibrierungslösung. </li></ol> 8. Datenerfassung <ol><li> Sobald die Kalibrierung durchgeführt wurde, zu erwerben Beispieldaten 4-5 zufällig ausgewählten Feldern mit 15-20 Zellen pro Feld. Zwei Bilder werden für jedes Feld erhalten: eine für HPTS Kennzeichnung (Vesikel) und die zweite für SNARF-1-Kennzeichnung (Cytosol). </li><li> Hintergrundfluoreszenz (extrazellulären Bereich) ist digital von Fluoreszenzbilder der beiden Sonden abgezogen. Beschriftete Vesikel und Cytosol von ihren jeweiligen gespeicherten Bilder werden mit Hilfe eines binären Maske ausgewählt. Für jeden pH-Messfühler sind Fluoreszenzintensitäten auf einer numerischen Skala (0-4,095 Graustufenskala) gemessen. Eine mittlere Intensität aller Pixel in der Organelle oder Zytoplasma wird berechnet und die Werte werden verwendet, um Fluoreszenz-Verhältnisse zu berechnen. </li></ol> 9. Datenanalyse <ol><li> Verschiedene Software-Pakete sind für die Datenanalyse im Handel erhältlich. DhFluoView Software. </li><li> In einer Tabellenkalkulation, Datensatzwerte und berechnen Fluoreszenzverhältnisse wie folgt: Im Fall von HPTS, R = (F 458 nm / F 405 nm) und, im Fall von SNARF-1, R = (F 644 nm / F 584 nm ). </li><li> Zeichnen Sie die Daten mit Hilfe wissenschaftlicher Grafik-Software wie GraphPad Prism. Das Grundstück sollte Fluoreszenzverhältnisse (Y-Achse) in Abhängigkeit von pH-Wert (X-Achse) zu porträtieren. Kurvenanpassung unter Verwendung nicht-linearer Regression gelöst. </li><li> Sammeln Fluoreszenzintensitäten der Proben, wie in Schritt 8.2 beschrieben und berechnen Fluoreszenzverhältnisse. Verwenden Eichkurven-Verhältnisse in pH-Werte zu transformieren. </li></ol>

<ol>
	<li>Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+Homeostasis+of+cellular+organelles.">pH Homeostasis of cellular organelles.</a> News Physiol Sci. 17, 1-5 (2002).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nat Rev Cancer. 11, 671-677 (2011).</li><li>Hinton, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+a+subunit+isoforms+of+the+V-ATPase+in+pH+homeostasis+and+in+vitro+invasion+of+MDA-MB231+human+breast+cancer+cells.">Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells.</a> J Biol Chem. 284, 16400-16408 (2009).</li><li>Overly, C. C., Lee, K. D., Berthiaume, E., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+intraorganelle+pH+in+the+endosomal-lysosomal+pathway+in+neurons+by+using+ratiometric+imaging+with+pyranine.">Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.</a> Proc Natl Acad Sci U S A. 92, 3156-3160 (1995).</li><li>Drose, S., Altendorf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycins+and+concanamycins+as+inhibitors+of+V-ATPases+and+P-ATPases.">Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases.</a> J Exp Biol. 200, 1-8 (1997).</li><li>Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., Cala, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NHE1+inhibition+by+amiloride-+and+benzoylguanidine-type+compounds.+Inhibitor+binding+loci+deduced+from+chimeras+of+NHE1+homologues+with+endogenous+differences+in+inhibitor+sensitivity.">NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity.</a> J Biol Chem. 282, 19716-19727 (2007).</li><li>Alfonso, A., Cabado, A. G., Vieytes, M. R., Botana, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-pH+crosstalks+in+rat+mast+cells:+cytosolic+alkalinization,+but+not+intracellular+calcium+release,+is+a+sufficient+signal+for+degranulation.">Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation.</a> Br J Pharmacol. 130, 1809-1816 (2000).</li><li>Han, J., Burgess, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+intracellular+pH.">Fluorescent indicators for intracellular pH.</a> Chem Rev. 110, 2709-2728 (2010).</li><li>Giuliano, K. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+intracellular+pH+of+BALB/c-3T3+cells+using+the+fluorescence+of+pyranine.">Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine.</a> Anal Biochem. 167, 362-371 (1987).</li><li>Willoughby, D., Thomas, R., Schwiening, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+intracellular+pH+changes+on+resting+cytosolic+calcium+in+voltage-clamped+snail+neurones.">The effects of intracellular pH changes on resting cytosolic calcium in voltage-clamped snail neurones.</a> J Physiol. 530, 405-416 (2001).</li></ol>

No conflict of interest declared.

Quantitative pH-Messung in lebenden Zellen von verschiedenen zellulären Kompartimenten wird benötigt, um pH-Schwankungen in Zell-Antworten auf externen Herausforderungen genau zu messen. Allerdings ist eine der größten Hürden, die bleibt, um einfach und gezielt zellulären Kompartimenten. In diesem Zusammenhang haben einige Studien den Einsatz von HPTS als intrazellulärer pH-Indikator durch Elektroporation oder Mikroinjektion in Zellen eingebracht 8-10 hervorgehoben. Diese Techniken leiden unter schwerw...

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Solutions for calibration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">D-Glucose (dextrose)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Magnesium sulfate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">HEPES</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Calcium chloride dihydrate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-1612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Nigericin</td>
			<td style="width:136px;">Calbiochem</td>
			<td align="right" style="width:119px;">481990</td>
			<td>Toxic by inhalation or in contact with skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;">Keep protected from the light at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Culture dishes &#8709; 35mm</td>
			<td style="width:136px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH-sensing probes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">H-348</td>
			<td>Keep away from the light at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">5-(and-6)-Carboxy SNARF&#174;-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">C-1271</td>
			<td>Keep away from the light and humidity at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Dimethyl Sufoxide (DMSO)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">BP231-1&#160;</td>
			<td>Harmful</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium phosphate monobasic</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">S369-1</td>
			<td>115 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium phosphate monobasic</td>
			<td style="width:136px;">J.T Baker</td>
			<td style="width:119px;">3246-01</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Autoclave and adjust pH to 7.4</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">PBS 100X</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>50 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td>40,5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deoinized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH modulators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">&#160;Bafilomycin A1</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">B-1793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Ammonium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">A649-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous ph measurement in endocytic and cytosolic compartments in living cells using confocal microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Für die gleichzeitige Messung von pH-Wert sowohl in der intrazellulären und der endosomalen / lysosomalen Zellkompartimenten wurden die ratiometrische Fluoreszenz-pH-Messfühler SNARF-1 und HPTS verwendet. SNARF-1 ist mit dem Zytosol beschränkt wohin HPTS ermöglicht ratiometrische pH-Messung der endosomalen / lysosomalen Kompartiment. Zellbeladung mit HPTS für 16 Stunden ermöglicht seine selektive Lokalisierung in Endosomen / Lysosomen 4. Ein typisches Beispiel der Fluoreszenz von HT-1080-Zellen mit den...

Watch this Scientific Journal Video about Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy at JoVE.com

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Gleichzeitige pH-Messung in endozytischen und Cytosolic Fächer in lebenden Zellen mittels konfokaler Mikroskopie

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

simultaneous-ph-measurement-endocytic-cytosolic-compartments-living

Research

JoVE Journal

Biology

14.9K Aufrufe.  Université de Sherbrooke. Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells. 

Serie: Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Forschung

Biologie

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Messung von pH Vacuolar und Cytosolic<em&gt; In Vivo</em&gt; In Hefe Zellsuspensionen

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analyse Craniofacial Morphogenese in Zebrafisch Mit 4D konfokale Mikroskopie

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Klonale Verfolgung von hämatopoetischen Stamm-und Vorläuferzellen durch Fünf Fluoreszierende Proteine ​​mit Hilfe der konfokalen und Multiphotonen Mikroskopie Markierte

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Cytosolischen Calcium Messungen in Nierenepithelzellen mittels Durchflusszytometrie

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Simultane Messung von Mitochondriale Calcium und mitochondrialen Membranpotentials in lebenden Zellen durch Fluoreszenzmikroskopie

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Eine grafische Benutzeroberfläche für die softwaregestützte Verfolgung der Proteinkonzentration in dynamischen zellulären Vorsprüngen

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Probing mRNA Kinetics in Space and Time in Escherichia coli mit Zwei-Farben-Einmolekülfluoreszenz in Situ-Hybridisierung

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral 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 compartmentalized, and that compartmentalization contributes to signaling specificity within the cAMP signaling pathway. The development of F&#1255;rster resonance energy transfer (FRET) based biosensors has furthered the ability to measure and visualize cAMP signals in cells. However, these measurements are often confined to two spatial dimensions, which may result in misinterpretation of data. To date, there have been only very limited measurements of cAMP signals in three spatial dimensions (x, y, and z), due to the technical limitations in using FRET sensors that inherently exhibit low signal to noise ratio (SNR). In addition, traditional filter-based imaging approaches are often ineffective for accurate measurement of cAMP signals in localized subcellular regions due to a range of factors, including spectral crosstalk, limited signal strength, and autofluorescence. To overcome these limitations and allow FRET-based biosensors to be used with multiple fluorophores, we have developed hyperspectral FRET imaging and analysis approaches that provide spectral specificity for calculating FRET efficiencies and the ability to spectrally separate FRET signals from confounding autofluorescence and/or signals from additional fluorescent labels. Here, we present the methodology for implementing hyperspectral FRET imaging as well as the need to construct an appropriate spectral library that is neither undersampled nor oversampled to perform spectral unmixing. While we present this methodology for measurement of three-dimensional cAMP distributions in pulmonary microvascular endothelial cells (PMVECs), this methodology could be used to study spatial distributions of cAMP in a range of cell types.

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and &amp;lambda; Hyperspectral FRET Imaging and Analysis

Due to inherent low signal-to-noise ratio (SNR) of F&#1255;rster resonance energy transfer (FRET) based sensors, measurement of cAMP signals has been challenging, especially in three spatial dimensions. Here, we describe a hyperspectral FRET imaging and analysis methodology that allows measurement of cAMP distribution in three spatial dimensions.

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 ...