Ice bindande proteiner (IBPS), även kända som antifrysproteiner, inhiberar is tillväxt och är en lovande tillsatsmedel för användning vid kryokonservering av vävnader. Det viktigaste verktyget som används för att undersöka IBPS är nanoliter osmometer. Vi utvecklade ett hem utformad kylsteg monterad på ett optiskt mikroskop och kontrolleras med hjälp av en specialbyggd LabVIEW rutin. Den nanoliter osmometer beskrivs här manipulerade provtemperaturen i en ultra-känslig sätt.
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 °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 μm 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).
Detta arbete demonstrerar driften av en datorstyrd nanoliter osmometer som möjliggör noggranna mätningar av TH-aktiviteten med utomordentlig temperaturkontroll. I varje temperatur-känsligt system, måste oönskade temperaturgradienter undvikas. För att undvika temperaturgradienter i apparaten som presenteras här, skall testlösningen droppen placeras i mitten av ett hål i kopparskivan kylsteget (steg 2,7). Dessutom bör den enda kristallen vara i centrum av droppen snarare än nära kanterna (i de flesta fall kommer detta att ske spontant). Tidsberoendet beskrivna visar att kylningshastigheten kan påverka TH avläsningar. Sålunda föreslår vi inklusive en rapport av den tid under vilken kristallen exponerades för lösningen före kylning, liksom kylningshastigheten. Vi väntade typiskt 10 min före ramp ner temperaturen vid 0,01 ° C steg vardera 4 sek.
De LabVIEW-kontrollerade samarbeteoling etappen anpassad för användning med ett omvänt mikroskop som mikrofluidikanordningar kan termiskt manipuleras. Detta system underlättar att testa lösningen utbyte som inbegriper iskristaller och IBPS etiketten EGFP 9, 10, 16. LabVIEW-styrda systemet kan anpassas till en Clifton steg genom att ansluta 3040 temperaturregulator via en utsedd anpassning elektrisk krets. Ett sådant system används i Davies labbet 17. Den LabVIEW programvara och den utsedda anpassa elektrisk krets design för Clifton scenen finns tillgängliga på begäran.
Sammanfattningsvis beskriver vi en nanoliter osmometer som underlättar känsliga kontroll och manipulation av temperatur och graden av temperaturen öka och minska (med 0,002 ° C känslighet), samordnas med en video-gränssnitt via en LabVIEW rutin för realtidsanalys. Detta system kan utföra reproducerbara kurs-kontrollerade experiment som är important för att undersöka kinetiken för IBP interaktioner med is. Sådana experiment kan ta flera långa omdebatterade frågorna kring verkningsmekanismen för IBPS.
The authors have nothing to disclose.
Denna forskning stöddes av ISF, NSF och ERC. Vi vill tacka för teknisk hjälp med temperaturen steg från Randy Milford, Michael Koren, Doug Shafer, och Jeremy Dennison. Hjälp med mjukvaruutveckling tillhandahölls av Eller Chen, Di Xu, Rajesh Sannareddy och Sumit Bhattachary. Vi vill tacka våra medarbetare Prof. Peter L. Davies och Dr Laurie A. Graham för Mp IBP protein och hjälp diskussioner. Vi tackar också lab medlemmar Dr Maya Bar-Dolev, Yangzhong Qin, Dr Yeliz Celik, Dr Natalya Pertaya, Ortal Mizrahy och Shlomit kille för sin användarfeedback.
Name | Company | Catalog Number/model | Comments |
Immersion oil Type B | Cargille Laboratories | 16484 | |
Drierite | W.A. Hammond Drierite | 043063 2270g | |
Micro 90 cleaning solution | Cole-Parmer | EW-18100-11 | |
Capillary puller | Narishige | PB-7 | |
Glass capillary tubes | Brand GNBH | 7493 21 | 75 mm long, 1.15 diameter |
Temperature controller | Newport, Irvine, California, United States | Model 3040 | Model 3040 |
Light microscope | Olympus | Model BH2 | |
10x objective | Olympus | S Plan 10, 0.3, 160/0.17 | |
50x objective | Nikon | CF plan, 50X/0.55 EPI ELWD | |
CCD Camera | Provideo | CVC-140 | |
Tygon tubes | Saint-Gobain, Paris, France | Tygon Formulation S-50-HL Tubing | |
Glass syringe (2 ml) | Poulten-Graf, Wertheim, Germany | 7 10227 | |
GPIB-PCI card | National instruments, Austin, Texas, USA | 778032-01 | |
Video frame grabber IMAQ-PCI-1407 | National instruments, Austin, Texas, USA | 322156B-01 | |
LabVIEW System Design Software | National instruments, Austin, Texas, USA | Version 8 | |
DiVx Author software | DiVx LLC, San Diego, CA, USA |