Is proteiner (IBPs), også kendt som anti-fryseproteiner, inhiberer is vækst og er en lovende additiv til anvendelse ved kryopræservering af væv. Det vigtigste redskab til at undersøge IBPs er nanoliter osmometer. Vi udviklede et hjem designet køletrin monteret på et optisk mikroskop og styres ved hjælp af en specialbygget LabVIEW rutine. The nanoliter osmometer her beskrevne manipuleret prøvens temperatur i en ultra-følsom måde.
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).
Dette arbejde demonstrerer driften af en computer-styret nanoliter osmometer der muliggør nøjagtige målinger af TH aktivitet med ekstraordinær temperaturstyring. I enhver temperaturfølsom system skal uønskede temperaturgradienter undgås. For at undgå temperaturgradienter i apparatet præsenteres her, skal testopløsningen dråbe anbringes i midten af et hul i kobber disc køletrin (trin 2.7). Desuden bør enkelt krystal være i midten af dråben ikke i nærheden af kanterne (i de fleste tilfælde vil dette ske spontant). Den tid beskrevne afhængighed angiver, at afkølingshastigheden kan påvirke TH aflæsninger. Således foreslår vi bl.a. en rapport af den tid, hvor krystallen var udsat for opløsningen forud for afkøling, og afkølingshastigheden. Vi typisk ventede 10 minutter før nedkørsel, temperaturen ved 0,01 ° C trin hver 4 sek.
De LabVIEW-kontrollerede cooling fase blev tilpasset til anvendelse med et omvendt mikroskop, som mikrofluidenheder kan termisk manipuleres. Dette system letter udførelsen af løsning udveksle forsøg med iskrystaller og IBPs markeret med eGFP 9, 10, 16. LabVIEW-styret system, kan tilpasses til en Clifton trin ved at forbinde 3040 temperaturregulator via en udpeget tilpasning elektrisk kredsløb. Et sådant system drives i Davies lab 17. Den LabVIEW software og den udpegede tilpasning elektrisk kredsløb design til Clifton scenen er tilgængelige efter anmodning.
Afslutningsvis beskriver vi en nanoliter osmometer der letter følsomme kontrol og manipulation af temperatur og hastigheden af temperaturstigning og fald (med 0,002 ° C følsomhed), koordineret med en video-interface gennem en LabVIEW rutine for real-time analyse. Dette system kan udføre reproducerbare rate-kontrollerede forsøg, der er important for at undersøge kinetikken af IBP interaktioner med is. Sådanne eksperimenter kan behandle flere lange omdiskuterede emner omkring virkningsmekanismen for IBPs.
The authors have nothing to disclose.
Denne forskning blev støttet af ISF, NSF, og ERC. Vi vil gerne anerkende teknisk hjælp med temperatur etape fra Randy Milford, Michael Koren, Doug Shafer, og Jeremy Dennison. Assistance med softwareudvikling blev leveret af Or Chen, Di Xu, Rajesh Sannareddy, og Sumit Bhattachary. Vi vil gerne takke vores samarbejdspartnere Prof. Peter L. Davies og Dr. Laurie A. Graham for Mp IBP protein og personer diskussioner. Vi takker også lab medlemmer Dr. Maya Bar-Dolev, Yangzhong Qin, Dr. Yeliz Celik, Dr. Natalya Pertaya, Ortal Mizrahy, og Shlomit Guy for deres brugerfeedback.
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 |