Buz Bağlayıcı Protein İncelemelerde LabVIEW çalıştırılan Roman nanolitre Ozmometre
Summary
Ayrıca antifriz proteinleri olarak bilinen buz bağlayıcı protein (IBPs), buz büyümesini engelleyen ve doku dondurma kullanım için umut verici bir katkı maddesi vardır. IBPs araştırmak için kullanılan başlıca araç nanolitre Osmometer olduğunu. Biz bir optik mikroskop üzerine monte edilmiş bir ev tasarlanmış soğutma aşamasında geliştirilen ve özel olarak geliştirilmiş bir LabVIEW rutin kullanılarak kontrol. Nanolitre Osmometer ultra-hassas bir şekilde numune sıcaklık manipüle burada açıklanan.
Abstract
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).
Protocol
Representative Results
Discussion
Bu çalışma olağanüstü ısı kontrolü ile TH aktivitesinin doğru ölçümleri mümkün kılar bir bilgisayar kontrollü nanolitre Ozmometre çalışmasını gösterir. Herhangi bir sıcaklık duyarlı sisteminde, istenmeyen sıcaklık değişimleri kaçınılmalıdır. Burada sunulan cihaz içinde sıcaklık gradyanları önlemek için, test çözeltisi damla bakır disk soğuma evresi (adım 2.7) içinde bir deliğin merkezi olarak konumlandırılmış olmalıdır. Ayrıca, tek bir kristal damlacık merkezinde ziyade kenarlarına yakın olmalıdır (çoğu durumda, bu kendiliğinden gerçekleşir). Açıklandığı zaman bağımlılığı soğutma hızı TH okumalarını etkiler olabileceğini gösterir. Böylece, kristal soğutma önce çözelti maruz kaldığı süreyi bir rapor, hem de soğutma hızı dahil olmak üzere göstermektedir. Biz genellikle 0.01 sıcaklık aşağı Ramping önce 10 dakika bekledi ° C her 4 sn adımlar.
LabVIEW kontrollü işbirliğioling aşama microfluidic cihazlar termal olarak manipüle edilebilir, üzerinde bir ters mikroskop ile kullanım için adapte edilmiştir. Bu sistem eGFP, 9, 10, 16 ile etiketlendi buz kristalleri ve IBPs içeren çözelti değiş tokuş deneylerin yapılması kolaylaştırır. LabVIEW kontrollü sistem belirli bir uyum elektrik devresi yoluyla 3.040 sıcaklık kontrol bağlayarak bir Clifton aşama için adapte edilebilir. Böyle bir sistem, Davies laboratuvar 17 işletilmektedir. LabVIEW yazılım ve Clifton aşaması için belirlenen adapte elektrik devresi tasarımı istek üzerine mevcuttur.
Sonuç olarak, hassas kontrol ve manipülasyon sıcaklık ve gerçek zamanlı analiz için LabVIEW rutin yoluyla bir video arayüzü ile koordineli sıcaklık artış ve azalış oranı (0.002 ° C hassasiyet ile), kolaylaştıran bir nanolitre Osmometer açıklar. Bu sistem importan vardır tekrarlanabilir hızı kontrollü deneyler gerçekleştirebilirsinizbuz ile IBP etkileşimlerinin kinetik araştırmak için t. Bu tür deneyler IBPs etki mekanizması çevreleyen birkaç uzun zamandır tartışılan sorunları ele alabilir.
Disclosures
The authors have nothing to disclose.
Acknowledgements
Bu araştırma ISF, NSF ve ERC tarafından desteklenmiştir. Biz Randy Milford, Michael Koren, Doug Shafer, ve Jeremy Dennison sıcaklık kademesi ile teknik yardım kabul etmek istiyorum. Yazılım geliştirme ile Yardımlaşma Veya Chen, Di Xu, Rajesh Sannareddy ve Sumit Bhattachary tarafından sağlanmıştır. Biz Mp IBP protein ve yararlı tartışmalar için ortak Prof Peter L. Davies ve Dr Laurie A. Graham teşekkür etmek istiyorum. Biz de teşekkür laboratuar üyeleri Dr Maya Bar-Dolev, Yangzhong Qin, Dr Yeliz Çelik, Dr Natalya Pertaya, Ortal Mizrahy ve kendi kullanıcı geri bildirimleri için Shlomit Guy.
Materials
| 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 |
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