DOI: 10.1002/asia.201100105
Laser-Induced Crystallization and Crystal Growth
their ablation at the focal point, inducing local bubble for-mation, shockwave propagation, and convection flow. This phenomenon, called “laser micro tsunami” makes it possi-ble to trigger crystallization of molecules and proteins from their supersaturated solutions. Femtosecond laser ablation of a urea crystal in solution triggers the addition-al growth of a single daughter crystaddition-al. Intense continuous wave (CW) near infrared laser irradiation at the air/solu-tion interface of heavy-water amino acid soluair/solu-tions results in trapping of the clusters and evolves to crystallization.
concentration. Upon irradiation at the glass/solution inter-face, a millimeter-sized droplet is formed, and a single crystal is formed by shifting the irradiation position to the surface. Directional and selective crystal growth is also possible with laser trapping. Finally, characteristics of laser-induced crystallization and crystal growth are sum-marized.
Keywords: crystallization · crystal growth · nonlinear pro-cesses · polymorphism · proteins
1. Introduction
Since the laser was invented in 1960, it has been contribu-ting to the development of modern chemistry—particularly in molecular spectroscopy and photochemistry. Early studies started on isolated molecules and clusters in the gas phase or dilute solution, then shifted to molecular complexes and polymers, and now are being extended to supramolecules, colloids, and molecular solids. The development of utilizing lasers from homogeneous to inhomogeneous systems has led us to combine lasers with optical microscopes.[1–4]
Time-re-solved spectroscopy has been integrated into time- and space-resolved spectroscopy, and resolutions have been im-proved so much that systematic studies have started in single molecular spectroscopy and are opening potential ap-plications in the life sciences.
Spectroscopy and photochemistry under a microscope usually need high intensity excitation, which is well known
in single molecule spectroscopy. In order to detect signals from small targets at a focal point, it is necessary to increase the excitation intensity, leading to various nonlinear photo-physical and photochemical processes; such as multiphoton ionization, excited-state annihilation, cyclic multiphoton ab-sorption, and so on. At higher intensities, laser ablation is induced, resulting in surface fragmentation. This is even pos-sible for transparent samples, as multiphoton absorption is so efficient using femtosecond laser excitation. Nowadays, laser ablation is popular for use in microfabrication tech-niques for various materials, and is being utilized in more complex solutions and biomedical systems. One characteris-tic of the ablation of solution is that it induces shockwave propagation, bubbling, and local convection flow, by which generates an impulsive force which pushes the surrounding micrometer-sized targets. This force can be used to manipu-late and to pattern microparticles and living cells in solution without damage. We are beginning to understand the vast potential of this manipulation and consider that this method is complementary to laser tweezers.[5]
This force is considered to generate a local area of high concentration transiently and triggers nucleation in supersa-turated solutions of proteins and molecules. Indeed, we suc-ceeded in the femtosecond laser-induced crystallization of lysozyme in 2002.[6] A supersaturated solution was
repeti-tively irradiated by a femtosecond Titanium:Sapphire laser and left in the dark, and then within a few days, several crys-tals had appeared. These systematic studies have been ex-tended from the viewpoint of the fundamental mechanism and application to complex proteins. This femtosecond ap-proach enables us to prepare crystals more speedily and to obtain crystals with better quality compared to those ob-tained from the conventional methods. Even membrane pro-teins, which had not been crystallized previously, will give a fruitful result.
In 2007, we demonstrated, for the first time, the laser trapping crystallization of glycine, which was realized by fo-cusing an intense CW laser beam.[7] Laser trapping is one
example of a laser application in science and technology that extends outside of a purely chemical application, and [a] Prof. Dr. T. Sugiyama
Graduate School of Materials Science Nara Institute of Science and Technology Takayama 8916-5, Ikoma, Nara 630-0192 (Japan) Fax: (+ 81) 743-72-6199
E-mail: [email protected] [b] Prof. Dr. T. Sugiyama
Instrument Technology Research Center National Applied Research Laboratories 20, R&D Rd. VI, Hsinchu Science Park Hsinchu 30076 (Taiwan)
Fax: (+ 886) 3-5773947
E-mail: [email protected] [c] Prof. Dr. H. Masuhara
Graduate School of Materials Science Nara Institute of Science and Technology Takayama 8916-5, Ikoma, Nara 630-0192 (Japan) Fax: (+ 81) 743-72-6199
E-mail: [email protected] [d] Prof. Dr. H. Masuhara
Department of Applied Chemistry and Institute of Molecular Science National Chiao Tung University
1001 Ta Hsueh Road, Hsinchu 30010 (Taiwan) Fax: (+ 886) 3-5712121 ext. 56593
has received much attention in bioscience for non-contact and non-destructive manipulation. This method does not in-volve photon absorption; namely, excited states of molecules are not generated. Most chemists have not paid attention to laser trapping. Therefore, we started exploratory studies on laser trapping in view of chemistry and soft materials. Ini-tially the targets of laser trapping were polymer spheres, droplets, silica gels, glass beads, catalysts, and so forth, with sizes in the visible micrometer order.[4]Scaling down to the
nanometer dimension followed in the middle of the 1990s. Polymers, micelles, J-aggregates, gold nanoparticles, and so forth, were trapped, aligned, and patterned on glass sub-strates in solution at room temperature.[8, 9]Their assembled
structures were prepared and analyzed, while trapping dy-namics were followed by single particle fluorescence spec-troscopy and fluorescence correlation specspec-troscopy. During these studies, we found that some organization is achieved in the optical trap, reflecting the properties of nanoparticles. This experience strongly stimulated the idea of forming crys-tals by laser trapping and was the starting point of research on laser trapping crystallization. Now, we are discovering more and more the conditions necessary for trapping crys-tallization and its great potential in modern crystal science. Garetz and Myerson conducted pioneering works in laser-induced crystallization, applying a nanosecond 1064 nm laser pulse. They induced nucleation of urea,[10, 11]
gly-cine,[12–14] lysozyme,[15] and l-histidine,[16] and explained the
phenomena in terms of a re-orientation of the molecules in a high-intensity laser field. This crystallization is nonphoto-chemical, as solvent or solute molecules are not excited by the laser. Recently, similar results have been shown with KCl,[17–19] suggesting a general applicability. On the other
hand, Okutsu and co-workers presented a series of papers on the photochemical crystallization of proteins.[20, 21]A
tryp-tophan residue in one protein photochemically reacts with another one in a different protein in solution, leading to a dimer, which gives nucleus for crystallization. In the present Review, we summarize femtosecond laser-induced crystalli-zation and laser trapping crystallicrystalli-zation methods, and de-scribe novel crystal growth phenomena introduced by laser irradiation. Furthermore, crystallization dynamics and mech-anisms are described in view of molecular interactions and the laser effect. Finally, the future perspective and expected outcomes are considered. All our laser-induced
crystalliza-tion behavior have been proposed, invented, and demon-strated on the basis of long studies in photochemistry, laser ablation, and laser trapping.
2. Crystallization and Crystal Growth by
Femtosecond Laser Irradiation
2.1. Transient Pressure Mechanism for Femtosecond Laser Ablation
It is generally considered that spectroscopic measurements and imaging are difficult during the laser ablation process, because ejected fragments may scatter monitoring and prob-ing lights. However, we found in the laser ablation of doped poly(methyl methacrylate) and microcrystalline Cu-phthalo-cyanine films, the optical conditions of the irradiated sample films are initially good to absorption spectroscopy. Indeed, we succeeded in measuring the femtosecond transient ab-sorption spectra of the latter film upon intense excitation. A decay of the Cu-phthalocyanine exciton band was observed in the time domain of 10 ps, and then was replaced by an ab-sorption spectrum with negative and positive bands. The latter bands were confirmed to be similar to those observed
Abstract in Japanese:
Teruki Sugiyama received his PhD from Nankai University, P.R. China in 2002. He was a Postdoctoral Fellow and ap-pointed Assistant Professor in Depart-ment of Applied Physics at Osaka Uni-versity in Japan under the supervision of Professor Hiroshi Masuhara, where he studied on preparation of organic nano-particles utilizing laser ablation technique in solution from 2002–2006. Then, he became a Researcher at Hamano Life Sci-ence Research Foundation in 2007, and then worked at Nara Institute of Science and Technology as a Research Associate Professor from 2008–2011, where he started his current research of a new topic in laser trapping crystallization of organic compounds and proteins. Now he is extending his research in Instrument Technology Research Center, National Applied Research Laboratories in Taiwan.
Hiroshi Masuhara graduated from Tohoku University in Sendai in 1966 and received his PhD in 1971 at Osaka Uni-versity, where his mentors were the late Professor Masao Koizumi and Professor Noboru Mataga, respectively. He worked in Mataga laboratory of Osaka University until 1984, and then had his own labora-tory in Kyoto Institute of Technology, Osaka University, and then Hamano Life Science Research Foundation, while he was also the director of ERATO Masu-hara Microphotoconversion Project, JST from 1988–1993. Now he is extending his research in Nara Institute of Science and Technology and National Chiao Tung University in Taiwan, where his groups are studying laser-induced crystallization of molecules and proteins and exploring new laser-induced phenomena in bio/nano systems by developing new laser–microscope methodologies.
in the temperature difference spectrum, so that we could es-timate the local transient temperature by comparing both spectra. The temperature elevation at a fluence of 100 mJ cm 2and at the ablation threshold were estimated to
be 250 K and only 100 K, respectively. These temperature elevations are too small to induce melting, decomposition, and rapid vaporization, but it should be noticed that the temperature elevation rate is extremely fast; 1013 K s 1. On
the other hand, surface roughening, arising from the onset of fragmentation, was confirmed to occur around 10 ns, which was made clear by surface light scattering imaging.
On the basis of these femtosecond transient absorption and imaging measurements, it was concluded that the elec-tronic excitation energy is converted to molecular and lat-tice vibrations in about 20 ps, whilst the attained local transi-ent temperature is insufficitransi-ent to induce morphological changes such as melting, sublimation, and decomposition. Thus, the photothermal mechanism cannot explain the pres-ent ablation, and we should focus our attpres-ention on the time lag between 20 ps and the morphological change. During this lag time, vigorous molecular motions lead to a local pressure increase and the force is directed in every direc-tion, as the irradiated volumes are surrounded by a non-irra-diated one, and thermal conduction cannot take place on the ps timescale. The mechanical stress accumulates in the film, and can be released by ejecting the upper part of the film while the lower layer remains on the substrate.
This ablation mechanism of transient pressure does not hold for nanosecond excitation, as during excitation, pulse enhanced molecular and lattice vibrations are transferred sufficiently to the surroundings. Namely, heat transfer from the irradiated area is brought about. Consequently, the pho-tothermal heating mechanism can be applied to nanosecond ablation. It should be noted again that femtosecond laser ablation is not photothermal but photomechanical, therefore the heating effect is suppressed. This is indeed a nice ad-vantage for molecular and protein crystallization, as thermal decomposition and denaturation can be avoided by applying femtosecond irradiation.[22]
2.2. Femtosecond Multiphoton Ablation in Solution Multiphoton laser ablation is easily induced by focusing a femtosecond laser pulse into water, leading to bubble for-mation. This is brought about at the focal point, pushing the surrounding water outside, which arises from the transient pressure effect in the photomechanical mechanism as de-scribed above. Consequently, a shockwave is generated and propagates with high speed, and leads to local convection around the focal point. When the bubble shrinks, counter-convection is accompanied. As a result, the mechanical force and water flow affect small objects located near the laser focus. We call this impulsive force a laser micro tsuna-mi, as it is induced locally and temporally in three-dimen-sional space in solution. These multiphoton laser excitation phenomena are well known, and its study and application have received much attention in view of medical purposes.
In our case, we focused the femtosecond laser near the target not at the target, and the generated laser micro tsuna-mi was applied in a series of experiments.[23, 24]The target is
not excited, no photophysical and no photochemical pro-cesses are induced, and no damage results, which is indeed an advantage of the laser micro tsunami. This approach has opened a new way to induce crystallization and to manipu-late living cells.
2.3. Molecular Crystallization by “Laser Micro Tsunami” This method was examined to prepare organic molecular crystals and proved to be useful. The 4-(dimethylamino)-N-methyl-4-stilbazolium tosylate crystal is an attractive materi-al for organic nonlinear optoelectronics because of its excel-lent optical properties, but an appreciably large quantity cannot be obtained easily. Its crystallization based on the femtosecond tsunami was demonstrated by us in 2005.[25]We
compared several crystallization experiments obtained with 30 000 shots of femtosecond laser irradiation at 1 kHz with those at 20 kHz and those without irradiation, and found that highly repetitive irradiation is most effective for nuclea-tion. X-ray diffraction analysis revealed that the quality of the obtained crystal is the same as that using the conven-tional method without irradiation.
A direct confirmation of crystallization dynamics by fem-tosecond irradiation was successfully obtained for a simple solution system: anthracene in cyclohexane. It is well known that crystallization is very easy and the crystal growth is con-siderably fast, so that it is possible to monitor the crystal growth within 1 s. A single shot femtosecond laser pulse with pulse energy above 3.1 mJ pulse 1 led to a sufficiently
supersaturated solution, when anthracene crystallization was induced at the vicinity of the laser focal point immediately after the irradiation. The threshold of crystallization was in agreement with that of the bubble formation. The evolution of the crystal is shown in Figure 1, where a bending filmlike crystal grew and changed to a normal shape. The bending form strongly suggests that the nucleation and initial growth started at the curved bubble surface, although the bubble itself was only observed at 100 ms after the irradiation.[26]
2.4. Lysozyme Crystallization by “Laser Micro Tsunami” The laser micro tsunami is considered an effective stimula-tion to form a nucleus in the supersaturated solustimula-tions of molecules and proteins. Both the propagating shockwave and the local convection assisting mass transfer increase the local concentration. As a result, the system, initially pre-pared in the supersaturated metastable state, possibly shifts to the crystallization area in the phase diagram as a result of the laser tsunami. This concept explains the reason why we succeeded in femtosecond laser-induced crystallization for lysozyme, a standard protein. Laser tsunami crystallization has now been extended to various proteins. Lysozyme crys-tallization is not difficult, but a more efficient cryscrys-tallization giving better quality, compared to the conventional method,
has been demonstrated. The pulse energy dependence of the crystallization is shown in Figure 2, representing the statisti-cal nature of the crystallization. Yoshikawa and co-workers reported that the effects of laser pulse energy, width, thresh-old values for crystallization, and bubbling were similar to each other, and that their values were lower for 200 fs pulse widths than those using 1.8 ps pulses.[27] These results
indi-cate that the crystallization is triggered by the bubbling. Laser tsunami and the following crystallization are complex
phenomena involving various processes, so the approach to clarify responsible parameters (in this case, the threshold) was crucial at the initial stage of our research.[28]
The questions we have received most frequently are whether or not multiphoton photochemistry of the protein is a key step for triggering crystallization. Namely, the photo-chemical reaction leading to a less soluble species possibly triggers the nucleation. By steady state illumination experi-ments, Okutsu and co-workers have reported that the photo-chemically formed tryptophan dimer is responsible for pro-tein crystallization. To exclude such a photochemical effect, we conducted an irradiation experiment shown in Figure 3.[23] A single droplet of lysozyme aqueous solution
was covered by paraffin oil and set on a microscope stage. The femtosecond laser pulse was introduced to the aqueous solution from the bottom, and, of course, the irradiation of the aqueous solution produced some bubbles and then some precipitants were observed, that is, the laser tsunami led to aggregation of proteins. In this experiment, we set the con-ditions to see visible mm-sized aggregates immediately after the irradiation, although the crystals came only after leaving the irradiated solution for a few hours. Next, a very intense femtosecond laser pulse was introduced to the oil phase and we confirmed that several bubbles were formed not only in the oil, but also in the aqueous solution. The bubbling in the oil phase should arise from the multiphoton excitation of oil, while the bubbles in the aqueous solution may be as-cribed to migrated bubbles formed in the oil phase or/and formed by the explosion of the interface between both phases. Lysozyme is insoluble in oil, so multiphoton excita-tion of lysozyme is not responsible for the phenomenon. Nonetheless after several seconds, precipitants appeared in the aqueous solution, which clearly indicates that the photo-chemical mechanism does not contribute to the crystalliza-tion.
To confirm another relation between bubbling and crystal-lization, we examined a spatial correlation between bubbling and crystallization in a viscous solution of hen egg-white ly-sozyme.[28] By adding polyethylene glycol, the viscosity
in-creased so that we expected that the formed bubbles would not diffuse out from the irradiated areas during the period necessary for crystal growth to form to a visible size.[28]
Ac-tually, 1 day after irradiation, lysozyme crystals were pre-pared and spatially associated with bubbles, with very few crystals found far from the bubbles. For example, we could observe a long-lasting bubble with an initial diameter of 120 mm. It shrunk to 80 mm in 1 hour and interestingly lyso-zyme crystals appeared at the surface of the bubble. This may indicate that the surface is a preferential field for the crystallization. The long-lasting bubbles are possibly com-posed of gas products from the photodissociation and photo-degradation of solute and solvent molecules owing to multi-photon absorption at the focal point. In the present supersa-turated solution, lysozyme and its clusters should be ad-sorbed at the surface of the formed bubble and their molec-ular ordering would lead to nucleation, and these nuclei undergo growth processes at the surface.
Figure 1. Microscopic images of crystallization process of anthracene upon single shot irradiation of 16.5 mJ pulse 1. Right side illustrations
rep-resent the generated crystals in left side photographs. Relative times are shown in the upper right of each frame; t1=the 1st frame when a crystal
was identified by eye after the bubble disappeared, t2=1 s and t3=2 s
after t1(Ref. [26]).
Figure 2. Pulse energy dependence of femtosecond laser-induced crystalli-zation in lysozyme solution, indicating the number of solutions that un-dergoes crystallization (Ref. [23]).
Similar approaches were adopted for clarifying crystalliza-tion processes of other proteins. Yoshikawa and co-workers studied femtosecond laser-induced crystallization of thauma-tin by adding agarose gel, and monitored the process over a time period ranging from ms to days.[27]Crystals and bubbles
were observed in the same area, which is ascribed to the suppressed diffusion of nuclei and bubbles. They also exam-ined fluorescence molecule-labeled lysozyme and reported that the shrinking of the bubble causes a local increase in the protein concentration.
This laser tsunami crystallization is useful to induce crys-tallization in solutions that have a low supersaturation degree. Murai and co-workers demonstrated that the use of a gel solution with agarose enhanced nucleation of egg white lysozyme crystals, and the crystallization was realized at a saturation degree that is 3 to 5 times lower than that re-quired without agarose and for spontaneous crystallization (without laser irradiation) with agarose.[29] Additionally,
fluorescence imaging of the labeled lysozyme revealed that the cavitation bubbles generate in the high concentration region around the focal point, which may trigger the nuclea-tion. Furthermore, the high concentration region remains for longer in the agarose system, so that the nucleation probability should be increased. The present laser tsunami crystallization method is improved by combining it with the solution-stirring technique,[30] which has received much
at-tention as a successful crystallization method for proteins.[31]
2.5. Directional Crystal Growth by Laser Ablation When nanoparticles are ejected from a molecular crystal in its supersaturated solution by femtosecond laser ablation, the nanoparticles and/or remaining holes in the crystal may become a seed for nucleation. Indeed, we found a quite novel phenomenon of laser-induced crystal growth in urea solution. One representative example is shown in Figure 4,
where a urea crystal is irradiated with a single 800 nm fem-tosecond pulse.[32] It is interesting to see that the daughter
crystal grew from the irradiated area, and the crystal shape is similar to a needle, which is characteristic of urea crystals (Figure 4 b). The daughter crystal tended to grow perpendic-ularly to the mother crystal, and the orientation of needle axis to the daughter crystal was independent of polarization of the laser pulse. At the higher energy irradiation (Fig-ure 4 b), several crystals were prepared near the irradiation area. The number of the daughters increased with both the laser energy and the number of pulses.
This growth was considered to be induced by etching and fragmentation, which were directly confirmed by AFM ob-servation. The ablation was done in air on a glass substrate and the obtained results are shown in Figure 5, where a hole etched in the urea crystal and fragments ejected on to the glass substrate are clearly identified. At higher laser fluence (Figure 5 b), many fragments, with sizes ranging from tens of Figure 3. Femtosecond laser-induced crystallization of lysozyme upon irradiating the laser into paraffin oil surrounding the lysozyme aqueous solution (Ref. [23]).
Figure 4. Subsequent crystal growth of urea from the irradiated area marked with an open circle by a single shot of an 800 nm pulse at (a) 0.12 and (b) 0.21 mJ pulse1(Ref. [32]).
nm to a few mm, were observed and they spatially dispersed on the substrate. These fragmentations are considered to be caused by the transient pressure effect as explained above, thus the destructive thermal effect should be suppressed in this femtosecond ablation.
The present laser-induced crystal growth is unconvention-al and considered useful in forming complex structures in supersaturated solutions, and the fragmentation results in nucleation and needle crystals growing perpendicular to the mother crystal. The subsequent processing gives a set of blocks as shown in Figure 6. Another idea is to use this phe-nomenon to generate a new single crystal when multicrystal-line materials or amorphous precipitates are formed unin-tentionally. Sometimes proteins and newly synthesized com-pounds are very expensive and/or difficult to obtain, and thus our approach will be used in relevant fields in near future.
3. Crystallization and Crystal Growth by Laser
Trapping
3.1. Laser Trapping Crystallization of Glycine Laser trapping is a well-known phenomenon of gathering macromolecules, micelles, nanoparticles, and molecular clus-ters in solution at room temperature.[33–36] The trapping
arises from the photon pressure of a focused laser beam, and the force increases along with the size and polarizability of the target object. Once the assembling is started at the focal spot, the polarization increases with the effective volume. Consequently, the formed assembly experiences a large trapping force, developing further trapping. Similarly, when laser trapping is applied to a supersaturated solution of an organic compound, it is expected that the solute
clus-ters formed in the solution are gathered to the focal spot, in-creasing the local solute concentration nonlinearly. Conse-quently crystallization is expected to be induced. For the first demonstration, we chose glycine as a solute for laser trapping induced crystallization. It is known for glycine that the dimer formation becomes prominent as the solution con-centration increases, and consequently the liquid-like solute clusters are formed by the link of the dimers through inter-molecular interactions.[37]D
2O was used as a solvent to
sup-press the heating in this experiment, since H2O has a
non-negligible absorption coefficient owing to the overtones of the OH vibration at 1064 nm.[38] In this glycine/D
2O
solu-tion, we succeeded in the first demonstration of laser trap-ping crystallization, as we named it, which is described in this paper.[7]
Figure 7 shows a series of crossed-Nicol images around the focal point during laser irradiation. Immediately after ir-radiation with a linear-polarized CW NIR laser beam with 1064 nm wavelength at the air/solution interface of the su-Figure 5. AFM images of the ablated fragments on a cover glass substrate
(right) and the corresponding etched area of a urea crystal (left) by one shot irradiation of an 800 nm laser pulse with (a) 0.031 mJ pulse1 and
(b) 0.056 mJ pulse1 through a 20 objective lens. Reproduced with
per-mission from Ref. [32].
Figure 6. a) Crystal patterning procedure for urea. The first pulse was ir-radiated to the open circle area numbered 1, the second pulse was to the circle 2, and so on. A single shot of the 800 nm femtosecond laser pulse with 0.12 mJ pulse 1was irradiated through a 10 objective lens. b) The
ladderlike spatial pattern of urea crystals obtained by successive single shot laser irradiation of the five open circle areas each under the same ir-radiation conditions. Reproduced with permission from Ref. [32].
Figure 7. Crossed-Nicol images of glycine crystallization during laser irra-diation (Ref. [7]).
persaturated solution thin film, only the laser reflection from the surface was observed as a small spot (Figure 7 a). After 16 s irradiation, one glycine crystal with a size of 10– 30 mm was clearly identified at the focal point by an EMCCD camera (Figure 7 b). Although we do not know how small a size we can detect with our set-up, the nuclea-tion was possibly induced at around 16 s. The formed crystal grew larger upon further irradiation while being trapped at the surface, and finally it measured about 50 mm (Figure 7 c). The laser power of 0.4 GW cm 2used in this experiment is
too small for one glycine molecule to be trapped stably at the focal spot.[39] Therefore, we suggest that the laser
trap-ping of the large liquid-like clusters, as described above, pro-vides the increase in local concentration followed by the nu-cleation. It is noteworthy that this laser trapping crystalliza-tion occurs only at the air/solucrystalliza-tion interface. This result indi-cates that both efficient trapping of the clusters at the sur-face and the re-orientation of glycine molecules contribute to the crystallization mechanism.
3.2. Control of Crystal Polymorph of Glycine We have also succeeded in controlling the crystal polymorph of glycine by tuning the laser power using laser trapping crystallization.[40] Glycine is one of the most representative
compounds to be studied for the polymorphic crystallization mechanism and process,[41–44]and it is known to have three
kinds of polymorph, namely of , b-, and g-form. The a-form is always produced by conventional crystallization methods,[45, 46]b-form is obtained by cooling a saturated
solu-tion with acetic acid,[47]and the g-form is prepared through
some experimental procedures of adding some additive salts or highly acidic/basic compounds.[41, 48, 49] Furthermore,
gly-cine requires a much higher supersaturation degree to crys-tallize into the g-form in solution.[50]However, such a higher
supersaturated solution is not easily prepared since concen-tration fluctuation in solution leading to the a-form is gener-ally unavoidable. The difficulty of g-form preparation is ex-pected to be overcome by utilizing this laser trapping tech-nique.
We investigated the poly-morph of crystals prepared under various laser powers be-tween 0.8 and 1.4 W. The ex-periment was done for 10 sam-ples at each condition. Note that only a single crystal was always formed at the focal point. FTIR measurement was carried out for all prepared crystals, and two kinds of spec-tra were obtained. All their vi-brational absorption peaks agree well with those of the a-or g-fa-orms,[51, 52] which was also
supported by single X-ray crys-tallographic analysis. Thus, we
successfully prepared g-glycine by this method, and found that the formation probability strongly depends on laser power as shown in Figure 8. The a-form was always ob-tained at a laser power less than 1.0 W, while the g-form became prominent with an increase in the power and a max-imum probability of 40 % was achieved at 1.3 W. However, when the power was set to 1.4 W, which is the maximum laser power in this experiment, the probability decreased to 20 %.
Here we discuss the preparation probability of g-glycine depending on laser power in terms of two effects: photon pressure and local temperature elevation at the focal spot. As illustrated in Figure 9 a, photon pressure should be in-duced by the interaction of a focused laser beam with gly-cine clusters, and the force is proportional to the laser power. Simultaneously, laser heating at the focal spot is in-duced, which mainly arises from the absorption of 1064 nm-photons by the glycine molecule itself. Since laser trapping Figure 8. Laser power dependence of probability of a g-glycine crystal prepared by laser trapping. Reproduced with permission from Ref. [40].
Figure 9. A bell-shaped curve of the multiplication of two effects; photon pressure and local temperature ele-vation. Reproduced with permission from Ref. [40].
of the clusters at the high power causes the increase in mo-lecular concentration at the focal spot, the absorption of NIR photons by the molecules becomes larger and larger, leading to further temperature elevation. As heating is always compensated by thermal dissipation to the surround-ings, the temperature elevation becomes more prominent at a higher power. Namely, the temperature elevation is nonli-nearly increased with the laser power. Next, we consider the changes in the supersaturation value at the focal spot, which depends on both photon pressure and local temperature ele-vation, as illustrated in Figure 9 b. Once the cluster is trapped, further trapping should be induced, and as a result, the supersaturation value at the focal spot nonlinearly in-creases with the laser power. Conversely, the local tempera-ture elevation, illustrated in Figure 9 a, nonlinearly decreases the supersaturation value. Consequently, the probability can be represented by the multiplication of these two compen-sating factors, giving a bell-shaped curve, as illustrated in Figure 9 c. Thus, a high degree of supersaturation is realized within a few minutes by this method, giving a single g-gly-cine crystal. We believe that this technique will become a standard crystallization method for crystal polymorph con-trol in the near future.
3.3. Dense-Liquid-Droplet Formation of Glycine Laser trapping crystallization of glycine is induced by focus-ing a laser beam at the air/solution interface. Thus, focal po-sition is one of the key factors for the crystallization. Indeed, when the focused laser beam is used to irradiate the solution, which is common in laser trapping experiments, a particle-like assembly of glycine with almost the same di-mensions as the spot size is confirmed although no crystalli-zation is realized.[33]During the investigation of photon
pres-suinduced phenomena depending on focal positions, re-cently, we successfully demonstrated the formation of a single millimeter-sized dense liquid droplet of glycine.[39]
This phenomenon is the first demonstration of a laser trap-ping behavior, which we summarize and describe here.
For all laser trapping experiments, laser heating and the accompanying phenomena should be always considered. Since the glycine molecule itself has a noteworthy absorp-tion coefficient at 1064 nm of the trapping laser, irradiaabsorp-tion causes the elevation in local temperature at the focal spot. When the laser beam is used to irradiate at the solution/ glass interface of the solution thin film, the generated heat is mainly transferred to the solution surface. Consequently the temperature distribution at the solution surface becomes non-uniform and results in local surface depression.[53, 54]
Ac-tually, on measuring the surface deformation with a surface displacement meter, the surface depression started immedi-ately after laser irradiation. The temporal change of the local surface height and the corresponding CCD images are shown in Figure 10 a and 10 b, respectively. Immediately after the irradiation, the surface linearly depressed, and fi-nally the solution thickness reached about 5 mm at around 18 s. It is interesting to see that the height suddenly
recov-ered by further irradiation. This amazing phenomenon is ob-served only for the glycine solution, never for a neat D2O
liquid film, which shows only the initial depression. After the surface height was elevated up to about 70 mm after 60 s irradiation, and spherical droplet formation gradually ensued around the focal spot. Eventually, at 200 s, the drop-let grew large up to a lateral diameter of 5 mm and height of about 150 mm, which was clearly identifiable with the naked eye.
The features of this amazing droplet are summarized as follows. First, the size is much larger than the focal spot of 1 mm, where the trapping force works. Since the trapping force is generated through the interaction of the laser beam with glycine clusters, the clusters outside the focal spot do not experience this force. Thus, we consider that the growth to mm-order for the droplet arises from the spontaneous molecular assembly, and is possibly assisted by molecular transfer toward the depression area, owing to convection. Second, the droplet surface height becomes higher than the initial thickness of the thin solution. Backscattering meas-urements with a He–Ne laser showed that the reflected light intensity increased nonlinearly with irradiation.[39] This
result indicates that the droplet has a higher molecular con-centration compared with the initial solution since the re-fractive index of glycine solution increases with concentra-Figure 10. a) Temporal change of the solution height upon focusing a laser beam at the glass/solution interface. b) CCD images around a focal spot simultaneously captured with (a). (1) and (2) in (b) correspond to those in (a). Reproduced with permission from Ref. [39].
tion.[55] Thus, the dense droplet prefers to conglobulate
be-cause of the high surface Gibbs energy, resulting in the higher surface height. Third, the droplet remains for several seconds, even after turning off the laser beam, and keeps the liquid-like phase prior to nucleation in spite of the high concentration. These characteristic features can be well ex-plained on the basis of liquid–liquid phase separation (LLPS). In 2007, He and co-workers attempted to induce LLPS of glycine in the aqueous solution by the rapid cooling method, however, a successful separation has never been re-alized so far.[56]Louchev and co-workers recently reported a
worthwhile theoretical result that stated mass transfer is en-hanced by a thermocapillary effect and as a result, effective-ly supplies solute molecules to the surface depression area, where the trapping force works.[57] These results support
that photon pressure is spatially integrated with the thermo-capillary effect, which induces a highly concentrated area that is sufficient to lead to LLPS. The droplet is also regard-ed as some kind of crystal precursor, since the nucleation is easily triggered within a few seconds by shifting the laser focus to the droplet surface. This success will give us valua-ble information to elucidate the early stages of the crystalli-zation process.
3.4. Control of Glycine Crystal Growth by Laser Trapping We have described above three notable phenomena of laser trapping of glycine clusters in solution: crystallization, poly-morph control, and dense liquid droplet formation. Next, we describe another interesting result of conducting the crystal growth by focusing the laser beam close to the spontaneous-ly generated crystal.[58]In order to explain the phenomenon
of this crystal growth, we give here Figures 11 and 12, where two samples A and B with one and three crystals around a focal spot are prepared, respectively. Figure 11 shows a series of crossed-Nicol images on irradiation for sample A— the white arrow tip denotes the focal spot. Note that the laser beam was irradiated at the solution/glass interface. Im-mediately after focusing the laser beam at a point 18 mm away from the crystal edge, crystal growth toward the focal spot was observed and the growth rate increased with irradi-ation time. When the edge of the growing crystal reached the focal spot and overlapped with it, the growth soon stopped. Then, by shifting the focal spot away from the edge again, it started re-growing. This is the first demonstration of crystal growth induced by laser trapping. For sample B, three spontaneously generated crystals could be observed around the focal spot (labeled I–III in Figure 12 a). When the focused laser beam was irradiated at the point between crystals I and II, as indicated by the white arrow, crystal I immediately started growing two-dimensionally. This growth behavior seemed to be quite different from that in sam-ple A, which showed one-dimensional growth. This may arise from an anisotropic property of the crystal growth, and crystal I in sample B probably has a two-dimensional crystal growth face. Simultaneously, crystal II conversely started shrinking from its upper side, which is located far from the
focal spot. Upon further laser irradiation, crystals I and II gradually became larger and smaller, respectively, and final-ly the latter was completefinal-ly dissolved. Incidentalfinal-ly, crysta-l III acrysta-lso grew scrysta-lightcrysta-ly toward the focacrysta-l spot by crysta-laser irradia-tion.
It is important to discuss the long range effect of the trap-ping force on crystal growth. Since the focal spot size is esti-mated to be only 1 mm, it is far too small to allow the force to reach the crystal. The key to explain this unique phenom-enon is to remember the result of the formation of the giant dense droplet, as described above. The formed droplet has a millimeter size, which means that the high concentration area is spread out over quite a large area compared to the focal spot. Therefore, the crystal can grow larger by use of the solutes in the high concentration area. On the other hand, the solution before irradiation should be under satura-tion since no spontaneous growth proceeded. The solutes used for the droplet formation should be compensated by the dissolution of other crystals in order to keep the chemi-cal equilibrium. These phenomena are much like Ostwald ripening, which arises from the difference of surface free energy. This successful demonstration of crystal growth will give us new trials of selective growth to a desired direction and crystal shape control by optimizing experimental condi-tions.
Figure 11. Crossed-Nicol images of laser trapping induced crystal growth of spontaneously generated glycine crystal. Reproduced with permission from Ref. [59].
Figure 12. Crossed-Nicol images of laser trapping induced crystal growth and dissolution of spontaneously generated glycine crystal. Reproduced with permission from Ref. [59].
4. Conclusions and Perspective
Femtosecond laser irradiation realizes new crystallization of molecules and proteins which are not available by conven-tional methods, offering fast and efficient crystallization that produces high quality crystals. It is considered that nuclea-tion takes place at the surface of laser-induced bubbles, which is followed by its growth. Laser trapping crystalliza-tion is made possible upon irradiacrystalliza-tion at an air/solucrystalliza-tion in-terface, and replaced by single droplet formation when irra-diated at a glass/solution interface. Crystal polymorph trol is achieved by tuning laser power, polarization, and con-centration, while crystallization has recently been made pos-sible even from unsaturated solutions.[59]
Both laser-induced crystallization methods are being de-veloped further to crystallize molecules, amino acids, and proteins, which are not obtainable using conventional tech-niques, and to control the crystal polymorph. In addition to the conventional conditions of concentration, solvent, and temperature, laser parameters, such as pulse width (or CW), wavelength, power, polarization, repetition rate, irradiation time, and so on, can be tuned for getting successful results. Practical preparation of new crystals is very much expected in many research fields, for which femtosecond crystalliza-tion will be fruitful. Elucidacrystalliza-tion of dynamics and mechanism of nucleation and crystal growth is important and will re-ceive much attention. In the case of laser trapping crystalli-zation, one single crystal is always fabricated in a focused position at an arbitrary time, which can be probed by spec-troscopic and imaging methods, so that the studies into crys-tallization dynamics and mechanism are very promising. Femtosecond laser crystallization and laser trapping crystal-lization are complementary to each other, and their concert-ed studies will open new horizons of crystal chemistry.
Acknowledgements
The present work is supported by a KAKENHI (S) grant (Grant-in-Aid for Scientific Research, No. 18106002) from the Japan Society for the Promotion of Science (JSPS) to H.M., the MOE-ATU Project (National Chiao Tung University) of the Ministry of Education, Taiwan, to H.M., the National Science Council of Taiwan (No. 0970027441) to H.M., a KAKENHI grant on Priority Areas “Strong Photon-Molecule Coupling Fields” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) (No. 21020022) to T.S., and a KAKENHI (C) grant (No. 20550136) to T.S.
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Received: February 4, 2011 Published online: June 30, 2011
