1. Pursuing the initial stages of crystal growth using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS) Takashi Sugiyama Miyasaka laboratory

2. Introduction Many studies have been done for decades to clarify the mechanism of crystallization. It is, however, its dynamics are too complicated to be understood in detail. Direct measurement of nucleation process requires a detection method of individual molecules moving freely in solution. It is still difficult even now. SolutionNano/micro crystalBulk crystalNucleation Initial stage

3. NsNs NcNc 0 ΔG c ΔG Thermodynamic background of crystal growth Aggregation size become larger than critical size. Crystal growth Critical size

4. Crystallization of colloidal particles Atomic force microscope High sensitivity High temporal resolution High spatial resolution Previous approaches for pursuing nucleation of crystals Laser scanning microscope molecular ordering dynamics of proteins at single molecule level on substrates ・・・・・・ High-sensitive photo detection methods have been developed recently, motivating researchers to pursue the crystal growth using them. Dynamic light scattering (DLS) Fluorescence correlation spectroscopy (FCS)

5. CONTENTS Principle of DLS and FCS Confocal setup Autocorrelation function (ACF) Pursuing crystal growth of naphthalene using DLS Pursuing protein nucleation using FCS Summary

6. Experimental setup for DLS and FCS Confocal optical setup Objective High spatial resolution (in particular, z-axis) can be achieved. Only fluorescent light from probe molecules or scattered light from crystals on the focal plane is detected. DLS: Scattering light from particles FCS: Fluorescent light from dyes Image plane Molecles/nanoparticles Laser light Pinhole High sensitive Photodetector (Single photon counting module) Sample solution

7. Autocorrelation function (ACF) ACF can be used to analyze many kinds of fluctuations. g(τ) : autocorrelation function (ACF) I(t) : signal intensity τ : delay time δ : fluctuations of intensity Fast fluctuationSlow fluctuation Slow decay of ACFFast decay of ACF

8. “Kinetics of the formation of organic molecular nanocrystals” Jack Adrian et al., Nanoletters, 1,141-143 (2001) Pursuing crystal growth of naphthalene using DLS Sample Ternary system: naphthalene/acetone/water Naphthalene/acetone solution Water Easy to control the solubility of naphthalene to the mixture solvent

9. Obtained ACF was fitted with Siegert relation : 0~1, experimental constant q : scattering vector magnitude : ACF Diffusion coefficient is determined. Stokes-Einstein equation η: viscosity T : temperature k : Boltzmann constant a : hydrodynamic radius Hydrodynamic radius can be calculated. : diffusion coeffcient D

10. Result The diffusion coefficient of the naphthalene nanocrystal decreased with time. Particle is monodisperse during each measurement. Sample (1) Naphthalene/Acttone/Water = 0.040/0.637/0.323 Sample (2) Naphthalene/Acttone/Water = 0.013/0.523/0.464 (1) (2) Sample (1) Growing process of naphthalene nanocrystals is pursued Incident beam:He-Ne laser(633 nm) Time(sec)

11. Other method is needed to pursue nucleation steps. Using DLS, time-evolution of the naphthalene crystal sizes (~100nm) under supersaturation could be pursued. In the DLS measurement, nucleation steps of the crystal cannot be observed because nucleation occurs faster than measurement time. NsNs NcNc ΔG c ΔG 0 Summary and assignment

12. “Screening crystallization conditions using fluorescence correlation spectroscopy” Maxim E. Kuil et al., Acta Cryst., D58, 1536-1541 (2002) Pursuing protein nucleation using FCS It is impossible to apply the FCS under high concentration of fluorescent probe, where fluctuation of fluorescent light is too small. Small amount of fluorescent labeled proteins are added to the solutions of unlabeled ones Possible to pursue nucleus (clusters) using FCS Labeled protein Unlabeled protein Supersaturated solution ・・・ High molecular concentration Free diffusionCluster

13. Sample : Lysozyme Protein Dye for labelling : Cy5 succinimidyl ester Cy5 labelled proteins were prepared. (Label ratio : 0.3~1.6 per protein) Cy5-labelled protein is homogeneously incorporated, suggesting labeled proteins affect their crystallization little. Concentration of labelled protein : ~5nM

14. Solubility change of proteins in adding electrolyte Effect of salting out Salting out constant :K s Ω σ :salting out term Λ :salting in term Ω σ depends on hydrophobic part of the surface and increasing rate of surface tension. Λ is independent of types of electrolyte and their concentration under high electrolyte concentration. In case of increase in electrolyte concentration… K s becomes large. ΩσΩσ large Λ not change

15. Model used for fitting: G(t): fluorescence intensity ACF N: number of particles T: fraction of fluorophores in triplet state τ t : triplet lifetime M: number of fluorescent component f i : fraction in i component S: structural parameter ACF Diffusion coefficient can be calculated. Relationship τ D (the average residence time) and D (diffusion coefficient) W xy is the radius of detection volume.

16. Diffusion rate became slow with increase in protein concentration correlation time t/μs Excitation light : He-Ne laser (633 nm) Viscosity rise of the solution due to an increase in the concentration of the protein Cluster formation of the protein Results

17. crystallizing 0.31M 1M no NaCl Hard sphere model Diffusion rate became slow and crystallizing occurred with increasing electrolyte concentration. Electrolyte concentration dependence (1) NaCl

18. When NaCl was added, diffusion rate became slow and crystallization occurred. Decrease of the volume for the proteins to move freely Protein cluster and/or nucleus formation The thickness change of electrical double layer From the experimental results Diffusion rate became slow although protein concentration was constant. No NaCl concentration dependence was observed on diffusion coefficient of lysozyme at low protein concentration. Protein cluster and/or nucleus formation was observed

19. Diffusion coefficient change of labeled lysozyme due to nucleation and/or association of the protein was pursued using FCS. The result suggests NsNs NcNc ΔG c ΔG 0 Calculation volume ratio (no NaCl : 1M NaCl )1 : 2.1 Existing probability no NaCl 0.31 M NaCl 1 M NaCl Critical nucleus Equilibrium shifts to the critical nucleus

20. 0.2M no (NH 4 ) 2 SO 4 1M Hard sphere model ・ Diffusion rate is independent of electrolyte concentration. ・ Crystallizing didn’t occur. (NH 4 ) 2 SO 4 Electrolyte concentration dependence (2) [Control experiment]

21. Summary Using FCS, the change of diffusion coefficient was observed when nucleation and/or association of the protein was occurred. The number of molecules inside critical nucleus has not been determined yet. Direct measurement of molecular motion will pave the way to further understandings of molecular nucleation.