Fig. 2.1 Schematic illustration of multiphoton absorption ... 18 Fig. 2.2 Absorption coefficient spectrum of water, together with the wavelength positions of the most widely used lasers ... 18 Fig. 2.3 Gibbs free energy versus specific volume of a pure substance for several temperatures starting at the saturation temperature up to the spinodal temperature ... 19 Fig. 2.4 Variation of vapor bubble nucleation rate with superheat temperature ... 19 Fig. 2.5 Schematic illustration of laser tsunami in water ... 19 Fig. 2.6 Plasma, shock wave, and cavitation bubble produced by Nd:YAG laser pulses of different duration and energy ... 20
xi
Fig. 2.7 Experimentally determined shock wave pressure ... 20
Fig. 2.8 Hydrophone signals measured at a distance of 10 mm from the emission center of the shock waves ... 20
Fig. 2.9 The diameter of the cavitation bubble in water as a function of time ... 21
Fig. 2.10 Spatially limited transient pressure generated by femtosecond laser can induce molecular nucleation in supersaturated solution ... 21
Fig. 2.11 Microscopic images of generated anthracene crystals (a) and a growth process of a bending film-like crystal created at the surface of a large laser-induced bubble (b-d) ... 22
Chapter 3 Experiment Fig. 3.1 Picture of mode-locked Ti:sapphire laser, Tsunami ... 24
Fig. 3.2 Absorption and emission spectra of Ti:sapphire ... 25
Fig. 3.3 The mode-locking principle in Tsunami ... 25
Fig. 3.4 Picture of Ti:sapphire regenerative amplifier system, Spitfire Pro ... 25
Fig. 3.5 The schematic illustration of CPA principle ... 26
Fig. 3.6 Laser light source and microscopic system for crystallization ... 27
Fig. 3.7 Picture of inverted microscope and other attachments ... 27
Fig. 3.8 Reflectance spectrum of the dichroic mirror used in the inverted microscope ... 27
Fig. 3.9 Transmittance spectrum of the objective lens used in the inverted microscope ... 28
Fig. 3.10 Illustration of the du Noüy ring method ... 30
Fig. 3.11 Illustration of the capillary method ... 30
Fig. 3.12 Picture of 1.5 ml glass bottle with 0.5 ml glycine solution inside ... 32
Fig. 3.13 Picture of crystallization plate ... 34
Chapter 4 Glycine Crystallization Fig. 4.1 The chemical structure of glycine ... 35
xii
Fig. 4.2 The shapes of (a) -polymorph and (b) -polymorph glycine crystals ... 36 Fig. 4.3 Pulse energy (J/pulse) dependence on glycine crystallization probability (%). ... 38 Fig. 4.4 The CCD image of cavitation bubble generation ... 39 Fig. 4.5 (a) Crystallization probability (%) and crystal morphology dependence on repetition rate (Hz). (b) Images obtained from CCD camera. (c) Picture taken by general camera. ... 40 Fig. 4.6 The crystallization probability (%) depending on the repetition rate (Hz) at the air/solution interface or glass/solution interface. ... 42 Fig. 4.7 The crystallization probability (%) depending on the distance from the air/solution interface (mm). ... 42 Fig. 4.8 The crystallization probability (%) depending on the repetition rate (Hz) at 3.0 M, 3.5 M and 4.0M ... 43 Fig. 4.9 The crystals obtained with different exposure times... 45 Fig. 4.10 (a) Raman image of obtained multiple crystals (b) Raman spectrum of obtained glycine crystal ... 46 Fig. 4.11 Mass spectra of standard sample and laser-induced crystal ... 47 Fig. 4.12 Glycine molecules in one unit cell. (Single crystal case) ... 49 Fig. 4.13 Illustration to explain the morphology difference because of the different repetition rates ... 50
Chapter 5 Application to Protein
Fig. 5.1 Three-dimensional conformation of hen egg white lysozyme (HEWL) ... 55 Fig. 5.2 The lysozyme crystal shape ... 55 Fig. 5.3 Crystallization probabilities and crystal images of lysozyme under spontaneous crystallization, irradiation inside the solution and irradiation at the air/solution interface (NaCl conc.: 10 mg/ml) ... 57 Fig. 5.4 Crystallization probabilities of lysozyme under spontaneous crystallization,
xiii
irradiation inside the solution and irradiation at the air/solution interface (NaCl conc.: 10, 20,
30 mg/ml) ... 58
Fig. 5.5 The reconstructed lysozyme molecular structures from the X-ray analysis data under different laser conditions (NaCl conc.: 10 mg/ml) ... 64
Fig. 5.6 Electron density distribution of one of -helices under different laser conditions (NaCl conc.: 10 mg/ml)... 64
Fig. 5.7 Electron density distribution of one of disulfide bonds under different laser conditions (NaCl conc.: 10 mg/ml)... 64
Fig. 5.8 Lysozyme concentration (mg/ml) dependence on surface tension ... 65
Fig. 5.9 NaCl concentration (mg/ml) dependence on surface tension ... 65
Fig. 5.10 Conformation changes of protein take place at interfaces... 66
Fig. 5.11 Salting out can keep lysozyme molecules refolding and can localize them even at the air/solution interface ... 67
xiv
List of Tables
Chapter 4 Glycine Crystallization
Table 4.1 The lattice constants and R-factors of glycine crystals with different morphologies…...………...49
Chapter 5 Application to Protein
Table 5.1 The lattice constants of lysozyme crystals under various experimental conditions………..………63
1
Chapter 1 Introduction
Laser light is regarded as a monochromatic light source showing high energy density
with high degree of spatial and temporal coherence. Laser beam can be focused to a very tiny
spot, and can be delivered at a large distance with extremely low divergence [1]. Therefore,
laser exhibits high potential and has been applied widely in diverse fields inclusive of physics,
chemistry, material science and so on. Undoubtedly, laser also plays an essential role in
various contemporary and interdisciplinary studies.
As one of laser applications, photoetching, an essential technique for microfabrications,
was first demonstrated when a pulsed ultraviolet excimer laser light with high enough
intensity was irradiated at a surface of organic polymer film and some morphological changes
were induced [2, 3]. Laser ablation was given to describe this phenomenon induced by pulse
laser irradiation at a solid material with intensity higher than a threshold [4]. If laser ablation
takes place in liquid or gaseous phase, the phenomenon is usually described as optical
breakdown. In fact, optical breakdown can be generated in water when focused femtosecond
laser pulses with high intensity are irradiated inside water [5]. The accompanying phase
2
transition of water from liquid to gas gives transient mechanical stress. The optical breakdown
has been employed for biological applications such as cellular surgery [6, 7], and the
consequent mechanical stress is applied to cellular arrangement or micropatterning [8] and
nanoparticle injection to single cells [9]. Nowadays, the application of optical breakdown has
been expanding to the studies on crystallization and crystal growth and has received much
attention [10-13].
Crystal is a solid material composed of atoms, molecules or even macromolecules which
are three-dimensionally arranged in a long-range order, and crystallization is the process from
atoms or molecules to a crystal. Making crystal is not only an important technique for
purification and separation, but also an indispensable process in determining crystal structures
both of inorganic solid-state materials and biological molecular crystals at atomic resolution
with X-ray diffraction analysis [14].
Among the biological macromolecules inclusive of proteins, nucleic acids and
carbohydrates, proteins constitute the largest group. Enzymes are the most diverse class of
proteins because nearly every chemical reaction in a cell requires a specific enzyme. In order
to understand cellular processes or design new structure-based drugs or pharmacological
agents, knowledge of the three-dimensional structure, functions and properties of enzymes
3
and other macromolecules are undoubtedly vital [15, 16]. X-ray diffraction of crystals and
nuclear magnetic resonance (NMR) are two widely-used techniques for the structural
determination of macromolecules at atomic resolution. While NMR does not require protein
crystals and provides more detailed information on the dynamics of the molecule in question,
it can be used only for biopolymers with molecular weight of less than 30,000. X-ray
crystallography can be applied to compound with molecular weight up to at least 106. For
many proteins, the difference is decisive in favor of X-ray diffraction [15]. Accordingly, a
major challenge to the full exploitation of X-ray diffraction technique is that the protein must
first be crystallized [16].
Protein crystallization is mainly a trial-and-error procedure in which protein is slowly
precipitated from its solution, so obtaining suitable single crystals is the least understood step
in the X-ray structural analysis of a protein [15]. As a matter of fact, there are still some
additional requirements for the crystallinity and size of the crystals if precise analysis results
are more likely to be obtained [17]. Moreover, since crystallization is a rate-limiting step from
single molecules to a crystal for some proteins such as membrane proteins which can be
attributed to inherent protein flexibility and to conformational inhomogeneity, it is also
necessary to shorten the crystallization time and elevate the crystallization possibility
simultaneously [18]. Therefore, laser-induced crystallization methods as novel crystallization
4
techniques have received much attention and have been proposed continually to try to satisfy
the present situation.
1.1 Introduction to conventional crystallization methods
Because amino acids are polar molecules, most of them can be dissolved well in water
rather than organic solvent. Accordingly, water is often used as solvent in crystallization. In an
aqueous solution at pH near isoelectric point, amino acids are in zwitterionic form, and their
zwitterionic property is still kept in crystal. For crystallization, general methods such as
cooling down saturated solution slowly, evaporating saturated solution slowly with adding
polar solvent such as alcohol, adding poor solvent into saturated solution, and adjusting pH to
lower the solubility of the solution are often used. If crystals with high quality are more likely
to be obtained, slow crystal growth at low supersaturated degree is preferred [14].
The history of protein crystal growth can be traced back about more than 150 years ago.
The first observed protein crystallization was published by Hünefeld in 1840, and the protein
was hemoglobin from earthworm. The crystals were obtained when the blood of an
earthworm was pressed between two slides of glass and allowed to dry very slowly. The
above description revealed that protein crystals can be obtained by controlling evaporation of
a concentrated protein solution. In other words, protein crystals can be produced by slow
5
dehydration which is one of the most acceptable concepts until now. Other conventional
methods such as temperature variation under otherwise constant conditions, the use of salt or
organic solvents as precipitating or crystallizating agents, and the use of metal ions were also
developed in the following decades [19].
The oldest crystallization technique is batch method which has been used for proteins
and nucleic acids crystallization for over 150 years and is regarded as one of the most reliable
crystallization methods. Batch method is attractive and convenient because of its inherent
simplicity and reproducibility. It requires nothing more than direct mixing of an unsaturated
protein solution with a precipitant solution and waiting for a period of time until spontaneous
nucleation commences. For instance, lysozyme, the most widely studied protein for protein
crystallization, is easily crystallized by batch method [19].
The most commonly used technique for protein crystallization is vapor diffusion
inclusive of hanging drop and sitting drop methods. Both of these two methods contain
sample solution and reservoir solution, and require a closed environment. At the early stage,
the sample solution has insufficient precipitate concentration for crystallization. After vapor
diffusion of solvent from sample solution to reservoir solution takes place and vice versa, the
precipitate concentration is slowly adjusted to an optimal degree for crystallization since the
6
Fig. 1.2 Schematic illustration of crystal formation from a disordered medium to a highly ordered phase [20].
amounts of solution are quite different between these two [14].
1.2 Introduction to crystallization process
Fig. 1.2 shows a schematic illustration of crystal formation from a disordered medium.
Compared with the final crystal phase, the initial disordered phase has higher free energy.
Crystallization starts from nucleation, in which a tiny embryo of a highly ordered phase with
lower free energy is formed. Nucleation seldom takes place spontaneously at unsaturated
concentration since it is relatively difficult for molecules to overcome some energy barriers
between these two phases. However, at high enough supersaturation, the energy barriers
decrease and the initial state becomes unstable, so that a tiny fluctuation can lead to the Fig. 1.1 Vapor diffusion for protein crystallization. (a) hanging drop
method (b) sitting drop method
(a) (b)
7
appearance of the new phase [20].
An illustration of phase separation during nucleation in detail is shown in Fig. 1.3. There
are two possible pathways from individual molecules to nucleation. When the size of
molecule accumulation increases to a critical size, the ordered nucleus forms and starts to
form crystal. On the other hand, dense liquid droplet or liquid-liquid phase separation
formation can be observed if the size of molecule accumulation continuously increases [20].
Fig. 1.3 Schematic illustration of the formation of ordered crystalline nuclei as a result of the superposition of a density and a structure fluctuations [20].
8
1.3 Light-induced crystallization
Light-induced crystallization can be realized with various light sources through different
mechanisms such as photochemical and nonphotochemical (or photophysical) processes. The
used light source can be Xe lamp, continuous wave laser or pulse laser at different
wavelengths ranged from UV region to NIR region.
1.3.1 Photochemical reaction-induced nucleation
Okutsu et al. have reported photochemical reaction-induced nucleation of anthracene
[21], hen egg-white lysozyme (HEWL) [22], thaumatin [23], and ribonuclease A (RNaseA)
[24]. In the lysozyme crystallization experiment, they exposed UV light (wavelength; 280,
300 and 400 nm) from Xe short arc lamp (wavelength; 200-800 nm, USHIO UXL-300D) to
Fig. 1.4 (a) Mechanism of the photochemically induced nucleation of lysozyme (b) enhancement of protein crystallization by a photochemical reaction [22].
9
supersaturated but metastable lysozyme solution and produced photochemical intermediate
(Fig. 1.4). Since the photoproduct has lower solubility in mother solution, it works as a
nucleus and triggers crystallization eventually [22]. Okutsu et al. gave a representative
demonstration of photochemical reaction-induced nucleation. However, it is not known for
sure how radical formation affects the purity of the obtained protein crystal.
1.3.2 Kerr effect-induced nucleation
Garetz et al. discovered that intense nanosecond NIR laser pulses (pulse duration; 20 ns,
repetition rate; 10 Hz, wavelength; 1.06 m) can induce nucleation of urea solution in 1996.
This phenomenon was named as nonphotochemical laser-induced nucleation (NPLIN).
Because it was observed that the initially forming needle-shaped crystals tend to be aligned
parallel to the electric field vector of the light, they proposed that the electric field-induced
realignment of urea molecules results in cluster formation and this mechanism is known as the
optical Kerr effect [25]. Nucleation of glycine [26] was also demonstrated, and the polymorph
of glycine was controlled by changing polarization of laser beam [27] or by additionally
applying strong DC electric field to enhance the optical Kerr effect [28].
10
1.3.3 Laser trapping crystallization
Continuous wave (CW) laser-induced crystallization has been developed very fast
recently. Fig. 1.5 shows the principle of laser trapping. When a highly focused laser beam
passes through sphere-like medium with a greater refractive index from surrounding, the
sphere-like medium acts like a weak positive lens and it will be moved toward the focal point
by a substantial net backward trapping force [29]. In laser trapping crystallization, tightly
focused CW NIR laser beam is employed to gather molecules or clusters, forming a high
concentration area at the focal spot, where the nucleation is eventually achieved. Since laser
trapping is actually a dynamic process against diffusion of trapped molecules or clusters from
the focal spot, in order to reach stable laser trapping condition, laser irradiation at a certain
point for a period of time is necessary. Therefore, selecting laser wavelength at which both of
solvent and solute have merely no absorption is quite important. Photons at NIR wavelength
Fig. 1.5 Diagram showing the ray optics of a spherical Mie particle trapped in water by the highly convergent light of a single-beam gradient force trap [29].
11
have lower energy and can be less absorbed by biological molecules or systems. However,
water as a common solvent is not applicable since it has vibrational absorption over 1000 nm
[30]. The absorption of water generates a serious problem in temperature elevation which
increases the solubility of solution, and crystallization becomes more difficult. In order to
solve this problem, it is necessary to replace water with deuterated water (heavy water) as
solvent in this crystallization method.
Sugiyama et al. has firstly demonstrated crystallization of glycine by an intense focused
CW-YVO4 laser beam (wavelength; 1064 nm) at the air/solution interface. It was observable
by a CCD camera that the glycine crystal grew from focus to a certain size within a few
seconds [31]. Furthermore, Rungsimanon et al. have investigated the method to control
glycine phase by tuning laser power. It was found that the competing result between photon
pressure and temperature elevation at a certain high laser power led to the probability increase
to find -polymorph glycine crystal, which is not available under ambient conditions [32]. On
the other hand, Yuyama et al. have demonstrated the preparation of a millimeter-scale dense
liquid droplet of glycine induced by laser trapping effect. After focusing a CW NIR laser
beam at the glass/solution interface of a thin film of its supersaturated heavy water solution
and forming the droplet, they found that crystallization starts immediately just after the focal
position is shifted to the air/solution interface. It is considered that the droplet formation is
12
possibly the early stage of the multistep crystallization process and plays an important role in
photon pressure-induced crystallization of glycine [33].
1.3.4 Molecular assembling induced by optical trapping
Tsuboi et al. reported that the aggregation of lysozyme as nucleus induced by optical
trapping in solution can trigger nucleation [34]. They also observed the assembling of amino
acids (glycine, proline, serine and alanine) induced by laser trapping in solution, and
considered that the observed glycine assembling may be experimentally verified as a
precursor for the crystal nucleus by Sugiyama et al. [35]. The differences between inducing
molecular aggregation as nucleus and forming a local high concentration area by trapping
laser are the focal position and the necessary time for observation. Molecular assembling is
induced in solution and takes several hours for observation, whereas laser trapping
crystallization can only be achieved at the air/solution interface and the crystal grows within
several seconds.
1.3.5 Femtosecond laser-induced crystallization
Femtosecond laser-induced crystallization of HEWL was firstly demonstrated in 2003 by
Hosokawa et al. [10]. Besides, urea [12], DAST (4-(dimethylamino)-N-methyl-4-stilbazolium
13
tosylate) [11], anthracene [13] and glucose isomerase (GI) [36] crystallizations were also
reported in following years. The mechanism will be introduced in detail in Chapter 2.
1.4 Motivation
In this work, we have focused on femtosecond laser-induced crystallization and tried to
clarify how laser parameters inclusive of pulse energy, repetition rate, focal position and
exposure time affect the crystallization probability, crystal morphology and crystalline phase.
Firstly, we demonstrated glycine crystallization since glycine crystal shows several
polymorphs and is well-studied in the field of crystal chemistry [14]. Then we applied our
obtained results to lysozyme crystallization since lysozyme is also a representative protein in
protein crystallography [19]. It is expected to sharpen present crystallization techniques with
optimizing laser parameters and simultaneously establish a remarkable milestone to study
molecular dynamics of femtosecond laser-induced crystallization under appropriate
conditions.
14
Chapter 2 Principle
The mechanism of femtosecond laser-induced crystallization is completely different
from other methods because only this technique triggers nucleation by multiphoton absorption
of solvent leading to optical breakdown and is less related with solute. Firstly, principle of
multiphoton absorption is explained with its schematic illustration in Fig. 2.1. Since the
energy of a single photon is not enough to excite a molecule from the ground state to a higher
electronic state, the molecule can be excited only by absorbing more than one photons
simultaneously and the energy difference between the two states is equal to the sum of the
energy of the photons. Figure 2.2 reveals that water has much higher absorption in UV region
due to electronic transition than in visible and NIR region due to vibrational overtone
absorption [37], so it supports that water molecule are excited by photons at 800 nm through
multiphoton absorption.
By focusing intense laser into water, multiphoton absorption of water occurs, and leads
to optical breakdown of water giving vigorous evaporation if the superheated condition is
satisfied. When the rate of volumetric energy deposition provided by laser irradiation is more
15
rapid than the rate of energy consumed by vaporization and normal boiling, the liquid water is
driven to a metastable superheated state. The liquid can remain metastable until the spinodal
temperature is reached. By viewing a plot of the Gibbs free energy versus specific volume at
ambient pressure as shown in Fig. 2.3, unique features of the spinodal temperature can be
appreciated. At the spinodal temperature, the equilibrium between saturated liquid state and
saturated vapor state becomes extremely unstable, so the liquid undergoes spinodal
decomposition, a spontaneous process by which a thermodynamically unstable liquid relaxes
decomposition, a spontaneous process by which a thermodynamically unstable liquid relaxes