Diese Arbeit wurde von der ISF, NSF und ERC unterstützt. Wir möchten technische Hilfe mit der Temperatur Etappe von Randy Milford, Michael Koren, Doug Shafer und Jeremy Dennison anzuerkennen. Unterstützung bei der Software-Entwicklung wurde von Oder Chen, Di Xu, Rajesh Sannareddy und Sumit Bhattachary vorgesehen. Wir möchten unsere Mitarbeiter Prof. Peter L. Davies und Dr. Laurie A. Graham für den Mp IBP Protein und hilfreiche Diskussionen. Wir danken auch lab Mitglieder Dr. Maya Bar-Dolev, Yangzhong Qin, Dr. Yeliz Celik, Dr. Natalya Pertaya, Ortal Mizrahy und Shlomit Guy für ihre Benutzer-Feedback.

0. Vorläufige Verfahren <ol><li> Glas zur Herstellung Injektionskapillare. Mit einer Kapillare Abzieher (Narishige, Tokyo, Japan), bereiten Sie einen scharfen Pipette mit einer feinen Öffnung von einer Glaskapillare Mikro-Schlauch (Brand GmbH, Wertheim, Deutschland). Die Größe der Öffnung sollte, indem Luft durch die Kapillare, um feine Blasen in sauberem Wasser zu erhalten verifiziert werden. Wenn die Kapillare geschlossen ist, dann kann es durch das Brechen der Kante zu öffnen. Dies kann durch Drücken oder Kratzen sie sanft gegen das Wasser mit Rohrwänden erreicht werden. Bereiten Sie die Kapillare, so dass die Öffnung blockiert ist, sondern annähernd ausreichend zu öffnen, um die Bildung von Blasen im Submillimeterbereich zu ermöglichen. </li><li> Copper Disc Reinigung. Beschallen die Kupfer-Discs für 10 min in 0,1% Micro-90 Seife (Cole-Parmer, Vernon Hills, Illinois, USA), dann waschen mit doppelt destilliertem Wasser. Führen Sie die Discs in einem Isopropanol (technische) Lösung und beschallen erneut für 10 min. FlosseVerbündeter, trocknen die Scheiben mit gefilterter Luft. Diese Reinigungsstufe ist entscheidend für die IBP Kontamination zwischen den Experimenten zu vermeiden. </li><li> Double-Layer-Deckglas Montage. Ein Deckglas Versammlung war bereit, für Probenbeobachtung ohne Kondensation von Feuchtigkeit auf dem Deckglas Oberfläche zu ermöglichen. Dies wurde durch Platzieren eines Drierite (WA Hammond Drierite, Xenia, Ohio, USA) Teilchen (2 mm Durchmesser) zwischen zwei Deckgläsern, die dann mit einer Heißklebepistole klebten erreicht. Diese Konfiguration verhindert Kondensation, die die Sicht versperren könnte, wenn die Probe auf niedrige Temperaturen abgekühlt und entfernt die Notwendigkeit, trockene Luft auf die Beobachtungsfenster zu blasen. </li></ol> Ein. Cooling Stage Set-up <ol><li> Verbinden Sie den Wasserfluss Einlass und Auslass der Kühlstufe bis 4 mm Innendurchmesser Tygon Röhren (Saint-Gobain, Paris, Frankreich), und schließen Sie den Wasserfluss Einlassrohr mit einer Wasserpumpe. </li><li> Verbinden einer 4 mm Innendurchmesser Tygon Röhre zu dem Einlaß der Kühlstufe to liefern trockene Luft. Die Luft wurde getrocknet unter Verwendung einer in-line Drierite Spalte. </li><li> Betreiben Sie die Luft-und Wasserpumpen. Beachten Sie, dass die Kühlelemente nicht ohne einen Kühlkörper betrieben werden. </li><li> Schalten Sie den Temperaturregler, Kamera und LabVIEW-Routine. </li></ol> 2. Probenvorbereitung <ol><li> Legen Sie eine 3-4 ul Tropfen Immersionsöl B (Cargille Labors, Cedar Grove, New Jersey, USA) auf der Rückseite eines 7 mm Durchmesser Kupfer Scheibe mit 500 um Löcher durch die Platte gebohrt. </li><li> Positionieren Sie den Kupfer-Disc auf der Kühlstufe mit dem Immersionsöl nach unten. </li><li> Verbinden des Kapillarrohrs (die stumpfe Kante) zu einer 0,7 mm Innendurchmesser Tygon Rohrs an dem anderen Ende mit einer 2-ml-Glas-Spritze (Poulten-Graf, Wertheim, Deutschland) verbunden ist. </li><li> Vor der Verwendung des Kapillarrohr, überprüfen Sie die kleine Öffnung der Kapillare, um sicherzustellen, dass die Öffnung eine geeignete Größe ist (siehe die vorläufigen Verfahren). </li><li> Langsam legen Sie die glass in die vorbereitete IBP Protein Probenröhrchen (2,4 uM Mp IBP-GFP in 20 mM CaCl 2 und 25 mM Tris-HCl bei pH 8, siehe Referenz 10 zur Herstellung Details) Kapillare und ziehen Sie das Glas Spritze, bis die Glaskapillare enthält 0,1 ul der Proteinlösung. </li><li> Beginnen Videoaufzeichnung über die LabVIEW-Software. </li><li> Das spitze Kante der Glaskapillare (das Protein enthaltenden Lösung) in einem der Löcher in der Scheibe auf dem Kupfer Kühlstufe. </li><li> Während der Beobachtung durch das Mikroskop (Olympus, Tokyo, Japan, 10fach Objektiv), sorgfältig durchdringen die Immersionsöl Schicht mit der Glaskapillare Spitze, und drücken Sie die Glasspritze (sehr fein), um eine kleine Menge (ca. 10 nl) des Proteins liefern Lösung, um eine 200 um Tropfen zu erzeugen. </li><li> Verschließen Sie das Loch in der Kühlstufe mit dem Double-Layer-Deckglas-Baugruppe (siehe die vorläufigen Procedures). </li></ol> 3. TH Activity Measurement <ol><li> Press die Kühlung Taste und stellen Sie die Temperatur auf -40 ° C. </li><li> Zunächst wird die Lösung Tropfen klar sein. Bei niedrigen Temperaturen, typischerweise im Bereich von -30 ° C bis -35 ° C, das Tröpfchen die Farbe ändert, was anzeigt, dass die Lösung eingefroren wurde. Unmittelbar nachdem die Probe eingefroren, erhöhen Sie die Temperatur langsam bis die Masse Eis beginnt zu schmelzen. Eine schrittweise Erhöhung der Temperatur ist erforderlich, um ein Überschwingen der Temperatur, die in dem vollständigen Schmelzen der Probe führen könnten, zu vermeiden. </li><li> Wechseln Sie zu einem 50x Objektiv und beginnen, um das Eis durch Einstellen der Temperatur schmelzen. Diese Anpassung ist interaktiv und die letzten Schritte sind typischerweise unter Verwendung von kleinen Schritten Temperatur von 0.002 ° C. Weiter zu schmelzen, bis ein Einkristall bleibt. Die endgültige Größe des Kristalls sollte etwa 10 um betragen. Die höchste Temperatur, bei der Schmelztemperatur aufgehört hat festgestellt wird der Schmelzpunkt zu sein und ist genau in der späteren Videoanalyse Stufe bestimmt. </li> &lt;li&gt; Stellen Sie die Temperatur um ein paar Hundertstel Grad Celsius unter dem Schmelzpunkt des Kristalls und beginnen einen Temperaturanstieg mit einer 10 min Verspätung. Passen Sie die Anstiegsrate wie gewünscht. Während dieser Zeit wird der Kristall mit den IBPs ausgesetzt werden. </li><li> Nach Abschluss der 10 min Einwirkzeit wird die Temperatur automatisch zu verringern, unter der Kontrolle des LabVIEW Routine. </li><li> Beachten Sie die Kristallform, wenn die Temperatur sinkt. An einem gewissen Punkt kann die plötzlichen Platzen der Eiskristall beobachtet werden. Die Temperatur, bei der dies der Fall ist, wie der Kristall Bersttemperatur vermerkt. </li><li> Verwenden Videoanalyse um die genaue Schmelzpunkt und die Burst Temperatur zu bestimmen. Erstens, indem Videoanalyse, finden Sie die genaue Schmelzpunkt. Erinnern, dass die höchste Temperatur, bei der das Schmelzen aufgehört hat bestimmt wird, der Schmelzpunkt ist. Dokumentieren Sie dieses Schmelzpunkt in einem Tabellenkalkulationsprogramm. Dann bestimmen die genaue crystal Bersttemperatur und dokumentieren diesen Wertsowie. Der Unterschied zwischen dem Schmelzpunkt und dem Gefrierpunkt oder Kristall Bersttemperatur ist die thermische Hysterese-Aktivität des IBP Lösung. </li></ol> 4. Die Messung der Zeit-abhängigen TH Activity <ol><li> Folgen dem Protokoll in Abschnitte von 3,1 bis 3,3 beschrieben, um einen Einkristall aus Eis herzustellen. </li><li> Nach der Bildung des Kristalls, die Verzögerungszeit der Rampe wie gewünscht, und schalten Sie auf die Rampe. </li><li> Die Temperatur wird mit einem festen Zinssatz zu verringern (nach der Betreiber-Anforderungen) automatisch, sobald die Rampe Verzögerungszeit abgelaufen ist. </li><li> Dokumentieren die Temperatur, bei der der Kristall-Burst auftritt. Berechnen der Belichtungszeit (die Zeit zwischen Kristallbildung und dem Kristall Burst). </li><li> Wiederholen des Versuchs für verschiedene Verzögerungszeiten und Plotten der TH-Aktivität in Abhängigkeit von der Belichtungszeit, um die Zeit-Abhängigkeit der TH-Aktivität zu bewerten. </li></ol>

<ol>
	<li>DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoproteins+as+biological+antifreeze+agents+in+antarctic+fishes.">Glycoproteins as biological antifreeze agents in antarctic fishes.</a> Science. 172, 1152-1155 (1971).</li><li>Worrall, D., Elias, L., Ashford, D., Smallwood, M., Sidebottom, C., Lillford, P., Telford, J., Holt, C., Bowles, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+carrot+leucine-rich-repeat+protein+that+inhibits+ice+recrystallization.">A carrot leucine-rich-repeat protein that inhibits ice recrystallization.</a> Science. 282, 115-117 (1998).</li><li>Raymond, J. A., Knight, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+binding,+recrystallization+inhibition,+and+cryoprotective+properties+of+ice-active+substances+associated+with+Antarctic+sea+ice+diatoms.">Ice binding, recrystallization inhibition, and cryoprotective properties of ice-active substances associated with Antarctic sea ice diatoms.</a> Cryobiology. 46, 174-181 (2003).</li><li>Tomchaney, A. P., Morris, J. P., Kang, S. H., Duman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+composition,+and+physical+properties+of+a+thermal+hysteresis+"antifreeze"+protein+from+larvae+of+the+beetle,+Tenebrio+molitor.">Purification, composition, and physical properties of a thermal hysteresis "antifreeze" protein from larvae of the beetle, Tenebrio molitor.</a> Biochemistry. 21, 716-721 (1982).</li><li>Kiko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+freeze+protection+in+a+sea-ice+crustacean+through+horizontal+gene+transfer.">Acquisition of freeze protection in a sea-ice crustacean through horizontal gene transfer.</a> Polar Biology. 33, 543-556 (2010).</li><li>Robinson, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+adaptation+in+Arctic+and+Antarctic+fungi.">Cold adaptation in Arctic and Antarctic fungi.</a> New Phytol. 151, 341-353 (2001).</li><li>Gilbert, J. A., Hill, P. J., Dodd, C. E., Laybourn-Parry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+antifreeze+protein+activity+in+Antarctic+lake+bacteria.">Demonstration of antifreeze protein activity in Antarctic lake bacteria.</a> Microbiology. 150, 171-180 (2004).</li><li>Raymond, J. A., DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+inhibition+as+a+mechanism+of+freezing+resistance+in+polar+fishes.">Adsorption inhibition as a mechanism of freezing resistance in polar fishes.</a> Proc. Natl. Acad. Sci. U.S.A. 74, 2589-2593 (1977).</li><li>Pertaya, N., Marshall, C. B., DiPrinzio, C. L., Wilen, L., Thomson, E. S., Wettlaufer, J. S., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy+evidence+for+quasi-permanent+attachment+of+antifreeze+proteins+to+ice+surfaces.">Fluorescence microscopy evidence for quasi-permanent attachment of antifreeze proteins to ice surfaces.</a> Biophys. J. 92, 3663-3673 (2007).</li><li>Celik, Y., Graham, L. A., Mok, Y. F., Bar, M., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superheating+of+ice+crystals+in+antifreeze+protein+solutions.">Superheating of ice crystals in antifreeze protein solutions.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5423-5428 (2010).</li><li>Gilbard, J. P., Farris, R. L., Santamaria, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolarity+of+tear+microvolumes+in+keratoconjunctivitis+sicca.">Osmolarity of tear microvolumes in keratoconjunctivitis sicca.</a> Arch. Ophthalmol. 96, 677-681 (1978).</li><li>Gilbert, J. A., Davies, P. L., Laybourn-Parry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperactive,+Ca2+-dependent+antifreeze+protein+in+an+Antarctic+bacterium.">A hyperactive, Ca2+-dependent antifreeze protein in an Antarctic bacterium.</a> FEMS Microbiol. Lett. 245, 67-72 (2005).</li><li>Soriano, J., Braslavsky, I., Xu, D., Krichevsky, O., Stavans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universality+of+persistence+exponents+in+two-dimensional+ostwald+ripening.">Universality of persistence exponents in two-dimensional ostwald ripening.</a> Phys. Rev. Lett. 103, (2009).</li><li>Tomczak, M. M., Marshall, C. B., Gilbert, J. A., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+facile+method+for+determining+ice+recrystallization+inhibition+by+antifreeze+proteins.">A facile method for determining ice recrystallization inhibition by antifreeze proteins.</a> Biochem. Bioph. Res. Co. 311, 1041-1046 (2003).</li><li>Knight, C. A., Hallett, J., Devries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solute+Effects+on+Ice+Recrystallization+-+an+Assessment+Technique.">Solute Effects on Ice Recrystallization - an Assessment Technique.</a> Cryobiology. 25, 55-60 (1988).</li><li>Celik, Y., Drori, R., Pertaya-Braun, N., Altan, A., Barton, T., Bar-Dolev, M., Groisman, A., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+experiments+reveal+that+antifreeze+proteins+bound+to+ice+crystals+suffice+to+prevent+their+growth.">Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 1309-1314 (2013).</li><li>Middleton, A. J., Marshall, C. B., Faucher, F., Bar-Dolev, M., Braslavsky, I., Campbell, R. L., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+protein+from+freeze-tolerant+grass+has+a+beta-roll+fold+with+an+irregularly+structured+ice-binding+site.">Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.</a> J. Mol. Biol. 416, 713-724 (2012).</li></ol>

Keine Interessenskonflikte erklärt.

Diese Arbeit zeigt, das den Betrieb eines computergesteuerten Nanoliter Osmometer, die genaue Messungen der TH-Aktivität ermöglicht außerordentlich Temperaturregelung. In jeder Temperatur-sensiblen System müssen unerwünschte Temperaturgradienten vermieden werden. Um Temperaturgradienten in der hier dargestellten Vorrichtung zu vermeiden, muss die Testlösung Tröpfchens in der Mitte ein Loch in der Kupferscheibe Kühlstufe (Schritt 2,7) positioniert werden. Außerdem sollte der Einkristall in der Mitte des Tropfens statt der Nähe der Ränder sein (in den meisten Fällen ist diese spontan geschehen). Die Zeitabhängigkeit beschrieben anzeigt, dass die Abkühlrate kann die TH Messwerte beeinflussen. Somit schlagen wir mit einem Bericht mit der Zeit, während der der Kristall zu der Lösung wurde vor dem Abkühlen ausgesetzt sowie der Abkühlgeschwindigkeit. Wir in der Regel warteten 10 Minuten vor dem Herunterfahren der Temperatur, bei 0,01 ° C Schritten zu je 4 sec. Die LabVIEW-gesteuerte ZusammenarbeitOling Stufe wurde für die Verwendung mit einem inversen Mikroskop, auf der mikrofluidischen Vorrichtungen thermisch manipuliert werden könnten angepasst. Dieses System erleichtert die Durchführung der Lösung Austausch Experimente mit Eiskristallen und IBPs mit eGFP 9, 10, 16 markiert. Die LabVIEW-gesteuerte System kann zu einer Clifton Bühne, indem Sie das 3040 Temperaturregler über ein bestimmtes Anpassung Stromkreis angepasst werden. Ein solches System ist im Labor Davies 17 betrieben. Die Software NI LabVIEW und der designierte Anpassung Stromkreis Entwurf für das Clifton Bühne sind auf Anfrage erhältlich. Abschließend beschreiben wir eine Nanoliter Osmometer, die das empfindliche Kontrolle und Manipulation der Temperatur und die Geschwindigkeit des Temperaturanstiegs Zunahme und Abnahme (mit 0,002 ° C Empfindlichkeit), mit einem Video-Schnittstelle über eine LabVIEW-Routine für die Echtzeit-Analyse koordiniert erleichtert. Dieses System kann reproduzierbaren Rate-kontrollierte Experimente, die importan sindt zur Untersuchung der Kinetik der IBP Interaktionen mit Eis. Solche Experimente angehen können mehrere lange diskutierte Fragen rund um den Wirkmechanismus von IBPs.

<table border="1"><tbody><tr><td> Name </td><td> Firma </td><td> Catalog Number / model </td><td> Kommentare </td></tr><tr><td> Immersionsöl Typ B </td><td> Cargille Laboratories </td><td> 16484 </td><td></td></tr><tr><td> Drierite </td><td> WA Hammond Drierite </td><td> 043063 2270g </td><td></td></tr><tr><td> Micro 90 Reinigungslösung </td><td> Cole-Parmer </td><td> <a href="http://www.coleparmer.com/Product/Cole_Parmer_small_sup_reg_sup_small_Micro_90_small_sup_reg_sup_small_cleaning_solution_case_of_four_4_Liter_bottles/EW-18100-11" target="_blank">EW-18.100-11</a> </td><td></td></tr><tr><td> Kapillar-Abzieher </td><td> Narishige </td><td> PB-7 </td><td></td></tr><tr><td> Glaskapillarrohre </td><td> Marke GnbH </td><td> 7493 21 </td><td> 75 mm lang, 1,15 Durchmesser </td></tr><tr><td> TemperaturRegler </td><td> Newport, Irvine, Kalifornien, USA </td><td> Modell 3040 </td><td> Modell 3040 </td></tr><tr><td> Lichtmikroskop </td><td> Olymp </td><td> Modell BH2 </td><td></td></tr><tr><td> Objektiv 10x </td><td> Olymp </td><td></td><td> S Plan 10, 0,3, 160/0.17 </td></tr><tr><td> 50x Ziel </td><td> Nikon </td><td></td><td> CF Plan, 50X/0.55 EPI ELWD </td></tr><tr><td> CCD-Kamera </td><td> Provideo </td><td> CVC-140 </td><td></td></tr><tr><td> Tygon Rohren </td><td> Saint-Gobain, Paris, Frankreich </td><td></td><td> Tygon Formulierung S-50-HL Tubing </td></tr><tr><td> Glasspritze (2 ml) </td><td> Poulten-Graf, Wertheim, Deutschland </td><td> 7 10227 </td><td></td></tr><tr><td> GPIB-PCI-Karte </td><td> National Instruments, Austin, Texas, USA </td><td> 778032-01 </td><td></td></tr><tr><td> Video Frame graBBER IMAQ-PCI-1407 </td><td> National Instruments, Austin, Texas, USA </td><td> 322156B-01 </td><td></td></tr><tr><td> LabVIEW System Design Software </td><td> National Instruments, Austin, Texas, USA </td><td> Version 8 </td><td></td></tr><tr><td> DivX Author Software </td><td> DiVx LLC, San Diego, CA, USA </td><td></td><td></td></tr></tbody></table>

labview-operated novel nanoliter osmometer for ice binding protein investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Messung der Zeitabhängigkeit TH Die LabVIEW-betriebenen Nanoliter Osmometer erleichtert die Durchführung von genauen TH-Aktivität Messungen. Die konstante Temperatur Reduktionsrate erlaubt die Messung der Zeitabhängigkeit TH. Die genaue Temperatursteuerung durch die Nanoliter Osmometer aktiviert war entscheidend für diese Experimente. Die Belichtungszeit von einem Eiskristall zu den IBPs in Lösung wird als die Zeitdauer von der Bildung des Kristalls (das Ende der Schmelzvorgang), bis der plötzliche Wachstum von Eis um den Kristall (Kristall Burst) definiert. Wir fanden, dass die Belichtungszeit der Eiskristalle zu den IBPs entscheidend die TH-Aktivität beeinflusst. Kurze Zeiträume IBP Exposition (ein paar Sekunden) produziert eine geringe TH-Aktivität in der Mp IBP-GFP-Lösung (2,4 uM) (Abbildung 5). Der TH-Aktivität mit IBP Belichtungszeit erhöht, bis sie ein Plateau bei 4 min IBP Exposition erreicht. Bei höheren IBP Konzentrationen, die Platteau wurde in kürzeren Zeiten erreicht. <img alt="Abbildung 1" src="/files/ftp_upload/4189/4189fig1.jpg" /> Abbildung 1. Schematische Darstellung IBPs Eis adsorbiert. Angenommen mit Genehmigung vom 10. <img alt="Abbildung 2" fo:src="/files/ftp_upload/4189/4189fig2highres.jpg" src="/files/ftp_upload/4189/4189fig2.jpg" /> Abbildung 2. Die Kühlstufe. A) zu Rohren auf dem Mikroskop verbunden. B) Ohne die obere Führung. C) Schematische Darstellung. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig2large.jpg" target="_blank"> </a><img alt="Abbildung 3" src="/files/ftp_upload/4189/4189fig3.jpg" /> Abbildung 3. Screenshot des LabVIEW-Schnittstelle. <a href="http://www.jove.com/files/ftp_upload/4189/4189fig3large.jpg" target="_blank">Cl</a>ick hier, um größere Zahl anzuzeigen. <img alt="Abbildung 4" fo:src="/files/ftp_upload/4189/4189fig4highres.jpg" src="/files/ftp_upload/4189/4189fig4.jpg" /> Abbildung 4. Temperaturstabilität graph. Der Temperaturregler wurde eingestellt, um die Temperatur 0,01 ° C alle 15 Sekunden zu senken. <img alt="Abbildung 5" fo:src="/files/ftp_upload/4189/4189fig5highres.jpg" src="/files/ftp_upload/4189/4189fig5.jpg" /> Abbildung 5. Mp IBP TH-Aktivität als Funktion der Eiskristall Belichtungszeit auf den IBPs. Jeder Zeitpunkt ist der Durchschnitt von 3-6 Experimenten.

Watch this Scientific Journal Video about LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations at JoVE.com

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Eis Bindungsproteine ​​(IBPs), auch als Frostschutzmittel Proteine ​​bekannt, hemmen Eiswachstum und sind eine vielversprechende Additiv zur Verwendung bei der Kryokonservierung von Gewebe. Das wichtigste Instrument zur IBPs zu untersuchen ist die Nanoliter Osmometer. Wir haben ein Haus entworfen Kühlstufe auf einem optischen Mikroskop montiert und kontrolliert über eine custom-built LabVIEW Routine. Die Nanoliter Osmometer hier beschriebenen manipulierten die Temperatur der Probe in einem ultra-sensitive Art und Weise.

LabVIEW-betriebenen Novel Nanoliter Osmometer für Ice Binding Protein Untersuchungen

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

labview-operated-novel-nanoliter-osmometer-for-ice-binding-protein

Research

JoVE Journal

Chemistry

20.4K Aufrufe.  The Hebrew University of Jerusalem. Eis Bindungsproteine ​​(IBPs), auch als Frostschutzmittel Proteine ​​bekannt, hemmen Eiswachstum und sind eine vielversprechende Additiv zur Verwendung bei der Kryokonservierung von Gewebe. Das wichtigste Instrument zur IBPs zu untersuchen ist die Nanoliter Osmometer. Wir haben ein Haus entworfen Kühlstufe auf einem optischen Mikroskop montiert und kontrolliert über eine custom-built LabVIEW Routine. Die Nanoliter Osmometer hier beschriebenen manipulierten die T...

Serie: LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

Oberflächenpassivierung für Einzelmolekül-Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

Forschung

Chemie

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Die kovalente Bindung von BMP-2 auf Oberflächen mit einem selbstorganisierten Monoschicht-Ansatz

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Bestimmung der eisbindenden Ebenen von Frostschutzproteinen durch Fluoreszenzbasierte Eisebene Affinität

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

Bio-Schicht-Interferometrie zur Messung der Kinetik der Protein-Protein-Interaktionen und allosterische Ligandeneffekte

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

Iridium (III) Lumineszierende Sonde zum Nachweis von Malaria-Protein Biomarker Histidin Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Wasser in Öl-Emulsionen: Ein neues System für die Montage wasserlösliche Chlorophyll-bindenden Proteine ​​mit hydrophober Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

Ein ELISA-basierte Bindung und Wettbewerbsverfahren zu schnell zu bestimmen, Ligand-Rezeptor-Wechselwirkungen

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE - Clear india geleluiert flüssige Fraktion Verleitung Elektrophorese

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

Untersuchungen über die Ga (III) -Komplexes von EOB-DTPA and Its<sup&gt; 68</sup&gt; Ga Radiolabeled Analog

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

Neuartige Techniken zur Beobachtung von Strukturdynamik von Photoresponsive Flüssigkristallen

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...