AFLUORESCENCE CORRELATION SPECTROSCOPY STUDY OF THE CRYOPROTECTIVE MECHANISM OF GLUCOSE ON HEMOCYANIN By Eric J. Hauger A THESIS Submitted to the faculty of the Graduate School of Creighton University in Partial Fulfillment of the Requirements for the degree of Master of Science in the Department of Physics. Omaha, NE May 6, 2014 Abstract Cryopreservation is the method of preserving biomaterials by cooling and storing them at very low temperatures. In order to prevent the damaging effects of cooling, cryoprotectants are used to inhibit ice formation. Common cryoprotectants used today include ethylene glycol, propylene glycol, dimethyl sulfoxide, glycerol, and sugars. However, the mechanism responsible for the effectiveness of these cryoprotectants is poorly understood on the molecular level. The water replacement model predicts that water molecules around the surfaces of proteins are replaced with sugar molecules, forming a protective layer against the denaturing ice formation. Under this scheme, one would expect an increase in the hydrodynamic radius with increasing sugar concentration. In order to test this hypothesis, two-photon fluorescence correlation spectroscopy (FCS) was used to measure the hydrodynamic radius of hemocyanin (Hc), an oxygen-carrying protein found in arthropods, in glucose solutions up to 20wt%. FCS found that the hydrody- namic radius was invariant with increasing glucose concentration. Dynamic light scattering (DLS) results verified the hydrodynamic radius of hemocyanin in the absence of glucose. Al- though this invariant trend seems to indicate that the water replacement hypothesis is invalid the expected glucose layer around the Hc is smaller than the error in the hydrodynamic radius mea- surements for FCS. The expected change in the hydrodynamic radius with an additional layer of glucose is 1nm, however, the FCS standard error is ±3.61nm. Therefore, the water replacement model cannot be confirmed nor refuted as a possible explanation for the cryoprotective effects of glucose on Hc. i Acknowledgements I would like to first thank the Creighton University Physics Department for all of the knowl- edge and guidance that they have provided over the years. With their help, my understanding of the world has grown deeply and I will be forever grateful for having them be part of my journey. To my advisor, Dr. Michael Nichols, thank you for your patience, for your kindness, and for inspiring me to pursue biophysics. Finally, I would like to thank my family, for their loving support and encouragement to achieve my goals. ii Contents 1 Cryopreservation and Motivations 1 1.1 Motivations . .1 1.1.1 Organ Preservation . .1 1.1.2 Human Reproductive Medicine . .1 1.1.3 Conservation of Endangered Species . .2 1.2 Modern Preserving Methods . .2 1.3 Protein Structure and Function . .3 1.3.1 Primary Structure . .3 1.3.2 Secondary Structure . .3 1.3.3 Tertiary Structure . .5 1.3.4 Quaternary Structure . .5 1.3.5 Loss of Protein Structure . .5 1.4 Cryopreservation Theories . .5 1.4.1 Vitrification Theory . .6 1.4.2 Water Replacement Theory . .7 1.5 Model Systems . .8 1.6 Previous Work . 10 1.7 Conclusion . 12 2 Optical Techniques to Evaluate the Water Replacement Model 13 2.1 Verifying the Water Replacement Model . 13 2.2 Fluorescence Correlation Spectroscopy (FCS) . 14 2.2.1 Multiphoton Excitation . 15 2.2.2 Diffusion . 18 2.2.3 Particle Fluctuations . 19 iii CONTENTS iv 2.2.4 FCS Design . 21 2.2.5 Filter Set . 22 2.2.6 FCS Correlation Function . 23 2.2.7 Photobleaching . 26 2.3 Dynamic Light Scattering (DLS) . 26 2.3.1 Rayleigh Scattering . 27 2.3.2 Interference . 28 2.3.3 DLS Correlation Function . 30 2.4 Conclusion . 31 3 Materials and Methods 33 3.1 Approach Overview . 33 3.2 Apparatus . 34 3.2.1 FCS System Preparation . 34 3.2.2 DLS System Preparation . 34 3.3 Methods . 34 3.3.1 Solution Preparation . 35 3.3.2 FCS Measurements . 36 3.3.3 DLS Measurements . 38 3.3.4 Data Analysis Procedure . 39 3.4 Conclusion . 40 4 Results and Discussion 41 4.1 FCS Results . 41 4.1.1 Power Dependence Study . 41 4.1.2 Detection Sensitivity Study . 42 4.1.3 Experimental Focal Volume . 44 4.1.4 Hemocyanin Studies . 47 4.2 DLS Results . 52 4.3 Evaluating the Water Replacement Hypothesis . 54 4.4 Conclusion . 57 Appendices CONTENTS v A Optics Protocols 65 A.1 FCS Laser Alignment . 65 A.2 Cleaning Optical Equipment . 65 B Solution Preparation 67 B.1 Hemocyanin . 67 B.2 10xCMFPBS . 69 B.3 1xCMFPBS . 69 B.4 20wt% glucose 1xCMFPBS . 69 List of Figures 1.1 Schematic of Generic Amino Acid Structure . .4 1.2 Formation of a Dipeptide . .4 1.3 Phase diagram for H2O and Glucose Solution. .6 1.4 Diagram of Vitrification Model . .7 1.5 Diagram of Water Replacement Model . .8 1.6 Fluorescent Amino Acid Spectra . .9 1.7 Diagram of Water Replacement Model . 11 2.1 Focal volume, intensity trace and FCS correlation function diagrams . 14 2.2 Schematic of 2PE process . 16 2.3 IPSF2 diagram . 17 2.4 Fluorescence intensity trace . 20 2.5 Schematic diagram of 2PE FCS setup . 21 2.6 Plot of System Transmission and L-tryptophan Fluorescence . 22 2.7 FCS example autocorrelation function . 25 2.8 Schematic diagram of DLS setup . 27 2.9 Relative scattering intensity of 5nm and 50nm particles . 29 2.10 Interference pattern for DLS . 29 2.11 DLS example autocorrelation function . 31 3.1 Screenshot for a FCS data run with 50nM Hc. 37 3.2 Screenshot for a DLS data run with 50nM Hc. 38 3.3 Autocorrelation graph showing timescales for various processes. 40 4.1 L-tryptophan power dependence study . 42 vi LIST OF FIGURES vii 4.2 Fluorescence Intensity vs Concentration plot for L-tryptophan, avidin spheres, and hemocyanin. 43 2 4.3 Plot of χυ vs lateral-to-axial 2PE focal volume aspect ratio. 45 4.4 Fits and residuals for 50nM Hc . 48 4.5 FCS autocorrelations of Hc in glucose . 49 4.6 Plot of τD vs concentration of glucose of 50nM Hc. 50 4.7 Viscosity plot of buffer solution vs the concentration of glucose. 50 4.8 FCS plot of RH vs the concentration of glucose of 50nM Hc. 51 4.9 DLS autocorrelations of Hc in solution at varying concentrations. 53 4.10 DLS plot of RH vs the concentration of Hc. 53 4.11 FCS histogram of Hc . 55 4.12 DLS histogram of Hc . 55 4.13 FCS and DLS plot of RH vs the concentration of glucose of 50nM Hc. 57 A.1 Photograph of the 2PE FCS system . 66 List of Tables 1.1 Amino Acid Fluorescence Data . .9 4.1 Average Hydrodynamic Radius of Hc from FCS . 52 4.2 Average Hydrodynamic Radius of Hc from DLS . 54 viii Chapter 1 Cryopreservation and Motivations 1.1 Motivations 1.1.1 Organ Preservation Every day 18 people die waiting for an organ transplant [1]. Organ preservation is consid- ered the “supply line for organ transplantation” [2]. The continuing shortage of donor organs and changes in donor demographics have propelled the need for achieving an optimal and effica- cious approach to organ preservation [3]. Currently the length of time an organ can be preserved depends on the organ but most cannot be kept viable for more than 24 hours [3]. If the viability of organs could be extended for months or even years then donor organs could be stored until patients in need, who best match the tissue being stored, could be prepped and placed into trans- plant surgery. Optimal storage would also help alleviate transplant complications due to delayed function rates of organs. Additionally, promising developments being made in tissue engineer- ing would increase our supply of available organs but ultimately effective storage techniques would be necessary [4]. Besides organ transplantation, developing and understanding optimal cryopreservation methods is an invaluable tool for human reproductive medicine and the con- servation of endangered species. Therefore, advancements made in cryopreservation have the potential to have great impacts in diverse areas of the scientific community. 1.1.2 Human Reproductive Medicine In the case of human reproductive medicine, techniques such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) along with advancements in female gamete acquisition have resulted in an increased need for sperm and oocyte cryopreservation methods to be devel- 1 CHAPTER 1. CRYOPRESERVATION AND MOTIVATIONS 2.