Selective Manipulation of L-Cysteine Crystal Polymorphs Using Focused Laser Beams

Selective Manipulation of L-Cysteine Crystal Polymorphs Using Focused Laser Beams | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Selective Manipulation of L-Cysteine Crystal Polymorphs Using Focused Laser Beams Teruki Sugiyama, Hozumi Takahashi, Hiroshi Yoshikawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5777300/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 May, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Abstract The selective manipulation of crystal polymorphism holds profound implications across diverse scientific and industrial fields, as distinct polymorphs exhibit unique physical and chemical properties. This study demonstrates selective polymorphic manipulation by laser trapping – a technique enabling contactless manipulation and condensation of matter at the nanometer-scale and micrometer-scale. L-cysteine, a ubiquitous amino acid employed in pharmaceuticals and food additives, was targeted. We reveal that continuous-wave laser irradiation yields single crystals of the metastable polymorph, whereas successive irradiation with high-repetition-rate femtosecond laser pulses induces poly-crystallization of the stable form. Crucially, by strategically alternating between these two laser modalities during crystal growth, we can open up new crystallization pathways, including the generation of single crystals of the stable phase. These findings underscore the significant potential of focused laser beams for precision polymorphic engineering, paving the way for the development of advanced materials with tailored properties. Physical sciences/Chemistry/Physical chemistry/Chemical physics Physical sciences/Materials science/Condensed-matter physics/Structure of solids and liquids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Crystal polymorphism —the ability of a material to exist in more than one crystalline form— has garnered significant attention across diverse scientific and industrial fields due to the distinct physical and chemical properties exhibited by different polymorphs. In the pharmaceutical sector, for instance, polymorphism profoundly influences critical drug characteristics such as solubility, stability, and bioavailability, all of which directly impact the drug's efficacy and safety. 1 , 2 Similarly, in materials science, polymorphism affects the mechanical, optical, and electronic properties of materials, influencing the performance of semiconductors, pigments, and energetic materials. 3 , 4 , 5 Consequently, developing effective strategies for the precise control of crystal polymorphs is crucial for designing crystalline materials with tailored properties. Traditionally, polymorphic control has been achieved by adjusting environmental parameters such as temperature, concentration, and solvent. 6 However, controlling the polymorphism of organic compounds is challenging due to the relatively weaker intermolecular forces driving their crystallization, such as van der Waals and hydrogen bonding. Even with a systematic screening of environmental parameters, the precise and reproducible preparation of the desired number of crystals and crystal polymorphs in organic compounds often remains difficult. Recent research has focused on the physical aspects of laser irradiation, such as electric field effects and heat generation, as a promising external perturbation for controlling the crystallization of various organic compounds. 7 , 8 , 9 , 10 Laser-based techniques such as Non-Photochemical Laser-Induced Nucleation (NPLIN) and laser ablation have shown potential in achieving polymorphic control, particularly in amino acids and pharmaceutical compounds. 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 Recent studies suggest that crystal nucleation and polymorphic selectivity through these methods are attributed to photothermally and/or photochemically produced bubbles, which locally increases solute concentrations and/or enhances heterogeneous nucleation. 18 , 19 , 20 Laser trapping, a technique that allows for the contactless manipulation and condensation of substances at the nanometer- and micrometer-scales, 21 , 22 , 23 , 24 has also emerged as a potential tool for the precise control of crystallization events. 25 Studies have demonstrated that focused irradiation with continuous-wave (CW) laser at the air/solution interface can induce crystal formation without bubble generation, and the polymorphs of the resulting crystals are highly dependent on laser parameters (laser intensity, polarization mode) as well as environmental conditions (e.g., initial concentration). 26 , 27 , 28 In terms of its mechanism, optical force, arising from the interaction between laser’s electric and solutes (theoretically treatment is summarized in Note 1), plays a crucial role in condensing the solutes at/around laser focus. 25 , 29 Interestingly, utilizing a high-repetition-rate femtosecond (HRR fs) laser instead of a CW laser can induce not only trapping effects but also bubble generation, 30 , 31 a phenomenon often observed in studies of NPLIN and laser ablation. 7 , 32 This bubble generation contributes to polymorphic control in several compounds. 30 , 31 While the condensation of solutes through trapping effects is likely essential for inducing crystal nucleation, 29 , 33 the critical factor governing polymorphic control remains elusive. Further investigation is necessary to achieve precise polymorphic manipulation. In this study, we investigated polymorph control in L-cysteine (L-Cys), an amino acid commonly used in pharmaceuticals and food additives, 34 by employing focused irradiation with CW and HRR fs lasers. L-Cys has also served as model compounds for polymorphic investigations. 35 , 36 , 37 , 38 Our findings revealed that CW laser trapping exclusively produced the α -form (metastable phase), while HRR fs laser trapping consistently yielded the β -form (stable phase). Additionally, we introduced a novel approach for single crystal fabrication by strategically switching between CW and HRR fs laser irradiation during the crystallization process. This technique allows for precise manipulation of the polymorphic outcome and paves the way for the controlled growth of L-Cys single crystals with desired properties. Results and Discussion Effects of Continuous-Wave Laser Irradiation on L-Cysteine Crystallization A near-infrared CW laser with a wavelength of 1064 nm was focused at the air/solution interface of an unsaturated L-Cys/D 2 O solution with a supersaturation value of 0.84 (also see Methods). Here, the saturation value ( SS ) was defined as the C / C e , where C and C e are the concentration of the sample and the saturated concentration (1.16 mol/kg at 25 ºC, Supplementary Note 2 and Supplementary Fig. 1), respectively. Figure 1 and Supplementary Movie S1 show the crystallization behavior of L-Cys under CW laser trapping. No discernible changes were observed immediately after laser irradiation ( t = 0 s). After several tens of minutes, a thin, rod-like crystal was formed at the laser focus ( t = 1165 s), subsequently developing into a hexagonal crystal with continued laser irradiation ( t ≥ 1191 s). The polymorphs of the generated L-Cys crystals were characterized by Raman spectroscopy (cf. Supplementary Note 3 and Supplementary Fig. 2). Throughout the crystallization process ( t ≥ 1165 s), Raman spectra confirmed the consistent presence of the α -form. Previous studies of polymorphism control using laser trapping have indicated the significant influence of laser parameters such as intensity and polarization on crystal polymorphs, crystallization probability, and induction time. 26 , 27 Therefore, we investigated the dependence of these parameters on the intensity of the linearly polarized CW laser. In this study, crystallization probability was defined as the ratio of successful trials, where crystal formation was observed within 40 minutes of laser irradiation, to the total number of trials. For example, if crystal formation was observed for 8 samples out of 10 trials, the crystallization probability would be 80%. To properly evaluate the tendency of the induction time, if crystallization was not observed within 40 minutes, the induction time was recorded as 40 minutes, indicating that the actual induction time is equal to or greater than 40 minutes. Figure 2 summarizes the results. Reliable L-Cys crystallization was observed at a laser intensity of 61 MW/cm 2 and higher, while crystallization was inconsistent at 37 MW/cm 2 . The induction time exhibited a decreasing trend with increasing laser intensity. This observation regarding the induction time was statically validated through the Kruskal-Wallis test, 39 which confirmed the significance of the difference ( H = 20, P = 0.0049%). Notably, despite variations in both crystallization probability and induction time, L-Cys crystals generated via CW laser trapping consistently exhibited the α -form. This exclusive formation of the α -form persisted across various conditions, including different polarization modes at 61 MW/cm 2 (Supplementary Fig. 3) and a systematic screening of solution concentrations from 0.98 to 1.35 mol/kg ( SS = 0.98–1.16), all of which consistently resulted in α -form crystallization. It is widely known that supersaturation plays a critical role in determining the polymorphic outcome of crystallization. As supersaturation is governed by both temperature and concentration, it is crucial to estimate the increase in both under CW laser trapping conditions to elucidate the mechanism underlying the exclusive formation of the metastable α -form. To observe the temporal evolution of concentration under laser trapping, we employed Raman spectroscopy to monitor the concentration dynamics of L-Cys. The L-Cys/D 2 O solution exhibits several peaks between 200 and 2000 cm − 1 (Supplementary Fig. 4a), and the intensity of some peaks (e.g., ~ 690, ~1430, and ~ 1870 cm − 1 ) was confirmed to be proportional to the L-Cys concentration. We tracked the concentration using the highest peak at ~ 1870 cm − 1 (calibration curve: Supplementary Fig. 4b), which allowed for rapid measurement of Raman intensity (5 seconds per spectrum). Figure 3 a shows the representative temporal evolution of Raman intensity at different laser intensities (37, 61, and 86 MW/cm 2 ). The intensity initially increased linearly with irradiation time, irrespective of the laser intensity. The intensity suddenly jumped upon crystallization, possibly facilitating the estimation of nucleation timing. The slope of the linear fitting curve before crystallization was (0.55 ± 0.12) × 10 − 3 a.u./s at 37 MW/cm 2 , (1.24 ± 0.18) × 10 − 3 a.u./s at 61 MW/cm 2 , and (2.28 ± 0.16) × 10 − 3 a.u./s at 86 MW/cm 2 . From the calibration curve (Supplementary Fig. 4b), the concentration increase rates were 0.6 ± 0.1 mmol/kg/s at 37 MW/cm 2 , 1.4 ± 0.2 mmol/kg/s at 61 MW/cm 2 , and 2.5 ± 0.2 mmol/kg/s at 86 MW/cm 2 (Fig. 3 b, left axis), with significant differences confirmed by the Kruskal-Wallis test ( H = 13, P = 0.19%). These differences explain the observed shorter induction time at higher laser intensities (Fig. 2 ). Unexpectedly, despite the varying rates of concentration increase, the actual concentration of L-Cys at the point of crystallization, as estimated from the Raman intensity at the intersection of the linear fitting curves before and after crystallization, was consistently around 2.5 mol/kg for all laser intensities, with no significant differences ( H = 1.1, P = 57%). This remarkable consistency reveals a critical threshold for supersaturation that must be attained to initiate the formation of the α -form, regardless of how quickly that supersaturation level is reached. Next, to accurately assess the supersaturation and its impact on the polymorphic outcome, we need to consider the effect of temperature elevation on L-Cys solubility. It is also essential to estimate the local temperature increase caused by laser irradiation, particularly since, for L-Cys, temperature influences which polymorph is formed (transition temperature: ≥ 32°C, cf. Supplementary Note 3). Therefore, we investigated the temperature elevation of the L-Cys/D 2 O solution upon CW laser irradiation. Based on the spectroscopic analysis (Supplementary Note 4 and Supplementary Fig. 5), the temperature elevation coefficient at the laser focus was determined to be 1.3°C/W. Consequently, in the laser intensity regime in this study, the maximum temperature elevation was estimated to be ~ 2°C (25°C → 27°C). Since this achieved temperature is considerably lower than the transition temperature (≥ 32°C), we concluded that temperature elevation from laser irradiation should not contribute to the exclusive crystallization of the α -form. Hence, we focused on calculating the supersaturation at the point of crystallization. According to a previous study, 40 a temperature elevation from 25°C to 27°C leads to a 4% increase in L-Cys solubility in H 2 O (see Supplementary Note 5). Assuming that the solubility in D 2 O also increases by 4%, the supersaturation at crystallization was estimated to be 2.5/(1.16 × 1.04) = 2.1. This value is significantly higher than that required for spontaneous crystallization ( SS ~ 1.2), indicating that laser trapping induces a highly supersaturated state prior to nucleation. It may seem surprising that laser trapping can induce such a high degree of supersaturation. Still, this phenomenon has been observed in previous studies on other amino acid and protein solutions. 29 , 33 , 41 , 42 , 43 It is important to note that nucleation frequency is governed by the magnitude of the activation energy required for nucleation, which is inversely proportional to the square of the difference in chemical potential between the solution and crystal and proportional to the cube of the surface free energy of the generating crystal. 44 , 45 Additionally, the surface free energy of the metastable phase is generally smaller than that of the stable phase due to the relationship between the enthalpy of the metastable phase and the stable phase. Therefore, under conditions where supersaturation is sufficiently high for both polymorphs, the activation energy required for nucleation of the metastable phase is smaller than that of the stable phase, making the metastable phase kinetically accessible. This could explain the exclusive crystallization of the metastable α -form upon CW laser trapping. Effects of high repetition-rate femtosecond laser on L-Cys crystallization. In the preceding section, we observed that CW laser irradiation consistently resulted in the crystallization of the α -form of L-Cys despite adjusting parameters such as laser intensity and solution concentration. This observation led us to hypothesize that alternative laser-based perturbations might be required to induce the crystallization of the β -form. To explore this possibility, we employed a focused HRR fs laser, which not only induces laser trapping but also causes additional phenomena such as laser ablation. 30 , 31 Fig. 4 and Movie S2 show the representative crystallization behavior of L-Cys under HRR fs laser trapping. This experiment also employed the unsaturated L-Cys/D 2 O solution with SS = 0.84. No apparent change was observed immediately after the laser irradiation. After several minutes of irradiation, bubbles were suddenly generated at the laser focus ( t = 1665 s). During bubble generation, small crystals (indicated by a red circle in the figure) were repeatedly produced and dissolved around the laser focus. Suddenly, rectangular crystals (indicated by green arrows in the figure) were yielded around the laser focus ( t = 1675 s). These rectangular crystals grew substantially with continued laser irradiation. When the growing crystals were trapped at the laser focus, they underwent ablation, resulting in poly-crystallization ( t = 1680 s). Surprisingly, Raman spectroscopy confirmed that these rectangle crystals correspond to the stable phase of β -form, which was never observed with CW laser trapping. Due to their limited size, we were unable to characterize the small crystals that formed prior to β-form crystallization using Raman spectroscopy. Nevertheless, we employed alternative methods to determine that these crystals were likely the α -form, as detailed in a later section. To examine the impact of HRR fs laser irradiation on the crystallization behavior of L-Cys, we investigated the dependence of eventual polymorphism, crystallization probability, and bubble generation timing on the intensity of the linearly polarized laser. The results are summarized in Fig. 5 . Crystals always appeared at 61 MW/cm 2 and 86 MW/cm 2 , while some trials failed at 37 MW/cm 2 . The bubble generation timing was significantly accelerated with increasing laser intensity ( H = 14, P = 0.08%). While such crystallization behavior is similar to that of the CW laser (Fig. 2 ), the HRR fs laser irradiation always produced the stable phase of β -form eventually. This result underscores that not only laser trapping but also additional phenomena characteristic of HRR fs laser contributed to the generation of β -form. We now discuss the crystallization and polymorphism mechanism of L-Cys by HRR fs laser irradiation. Unlike the case of CW laser irradiation, successive irradiation with HRR fs laser suddenly produces bubbles at the laser focus (Fig. 4 , t = 1665 s). Such bubble generation has also been observed in the experiments on HRR fs laser trapping-induced crystallization using other amino acid solutions. 30 , 31 This phenomenon can be explained as follows: the focused irradiation with a HRR fs laser induces a concentration increase at the laser focus due to its laser trapping effect. 46 , 47 As the concentration is increased, the multiphoton absorption by solutes is enhanced due to the considerably higher peak intensity of the HRR fs laser (≥ 3.8 TW/cm 2 ) compared to the CW laser (≤ 86 MW/cm 2 ). When the concentration reaches a certain level, the multiphoton absorption triggers laser ablation of the solution, producing bubbles. 48 These generated bubbles may correspond to cavitation bubbles that are often produced by irradiation with ultrashort laser pulses. 49 Indeed, it has been reported that bubble generation by laser ablation of solutions can induce crystal nucleation. 7 , 50 As for its mechanism, several studies have proposed that local concentration increase and/or heterogeneous nucleation at the bubble surface plays an important role in crystal nucleation by laser ablation. 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 The preferential nucleation of the α-form polymorph can be attributed to the localized increase in solute concentration induced by laser trapping and the subsequent generation of cavitation bubbles. This localized concentration increase leads to a high degree of supersaturation, favoring the nucleation of the α-form, which is characterized by lower surface free energy. As the α -form crystals grow, solute molecules are consumed, leading to a decrease in the supersaturation level within the vicinity of the laser focus. The decrease in supersaturation causes a shift in the dominant factor for nucleation, from surface free energy to chemical potential. Consequently, subsequent bubble generation events within this region of reduced supersaturation promote the nucleation of the β -form polymorph, which possesses a lower chemical potential and becomes thermodynamically favored under these conditions. This phenomenon exemplifies the delicate interplay between supersaturation, surface free energy, and chemical potential in determining the polymorphic outcome of crystallization processes. The initial dominance of surface free energy under high supersaturation conditions gives way to the dominance of chemical potential as supersaturation decreases, effectively dictating the transition from α-form to β -form nucleation. In fact, such a conversion from metastable polymorph to stable polymorph has also been observed in HRR fs laser trapping of L-phenylalanine. 30 Selective Manipulation of Polymorphs by Combination of CW Laser and HRR Fs Laser The results presented in the preceding sections demonstrate that the eventual polymorph of L-Cys can be controlled by selecting the appropriate laser irradiation mode: CW laser irradiation consistently yields the α -form, while HRR fs laser irradiation ultimately produces the β -form. Building upon these findings, we explored the possibility of achieving dynamic control over the eventual number of the crystals and the resulting polymorph by strategically combining CW and HRR fs laser irradiation. For this purpose, we designed an experiment where we switched between CW and HRR fs laser irradiation during crystal growth. As shown in the leftmost image in Fig. 6 , we first irradiated the HRR fs laser at 86 MW/cm 2 into the L-Cys solution until bubble generation was observed at the laser focus. Immediately confirming the bubble generation, we switched to CW laser irradiation. This change in irradiation mode led to the continued growth of the large single α -form crystal (~ 200 µm) without producing the β -form (the upper path in Fig. 6 and Supplementary Movie 3). This result indicates that switching to CW laser irradiation before β -form crystallization by the subsequent HRR fs laser irradiation can promote the growth of α -form crystals. This result also emphasizes that the small crystals observed before β -form crystallization are more likely to the α -form crystals. Furthermore, we confirmed that re-switching to HRR fs laser irradiation after sufficient growth of the α -form crystal eventually still led to the formation of the β -form (Supplementary Fig. 6 and Supplementary Movie 4). Specifically, re-switching to HRR fs laser initially induced etching of the crystals surface with generating bubbles. Approximately 2 minutes after re-switching to HRR fs laser, the β -form crystals suddenly appeared around the laser focus. This observation underscores the role of HRR fs laser irradiation in inducing β -form crystallization. The combination of CW laser and HRR fs laser also offers another advantage. Activating CW laser immediately after the crystallization of the β -form resulted in single crystal growth without poly-crystallization (the bottom path in Fig. 6 , Supplementary Movie 5). This precise control over single crystal growth is challenging to achieve with the HRR fs laser alone, as demonstrated in Fig. 4 . Thus, precise spatial and temporal manipulation of CW and HRR fs laser irradiation enables the preparation of single L-Cys crystals with the desired polymorph. This technique offers a new level of control over the crystallization and polymorphism process, allowing for the targeted production of specific polymorphs. Conclusions This study has delved into the selective manipulation of L-cysteine polymorphism, revealing the remarkable ability of focused laser irradiation to guide the polymorphism selectively. We have demonstrated that continuous-wave (CW) laser irradiation consistently yields the metastable α -form, while high repetition-rate femtosecond (HRR fs) laser irradiation favors the stable β -form. This intriguing divergence stems from the distinct mechanisms triggered by each laser type. CW laser irradiation generates a highly supersaturated state, promoting the rapid formation of the kinetically accessible α -form. Conversely, HRR fs laser irradiation induces bubble generation and localized concentration increases, ultimately tipping the scales toward the thermodynamically stable β -form. Taking this a step further, we achieved dynamic control over the crystallization pathway by strategically alternating between CW and HRR fs laser irradiation. This innovative approach, visually summarized in Fig. 7 , allowed us to achieve remarkable results in crystal engineering, such as growing large single crystals of the α -form and preventing polycrystallization of the β -form. The implications of this work extend far beyond L-cysteine. By harnessing the precise interplay of laser parameters and material responses, we can envision a future where we can manipulate crystal structures with unprecedented accuracy. This promises to revolutionize fields ranging from pharmaceuticals and electronics to optics and catalysis. Our findings illuminate a path towards a new era of materials design, where light serves as a sculptor of matter at the molecular level. Methods Sample preparation. L-cysteine (L-Cys, ≥ 99%, Sigma-Aldrich) was used as received without further purification. L-Cys crystals were completely dissolved in deuterium oxide (D 2 O) by stirring at 70°C for 15 minutes. D 2 O was chosen as the solvent instead of H 2 O to minimize temperature increases caused by the near-infrared CW laser irradiation. The temperature elevation coefficients (thermal absorption coefficients) at 1064 nm are reportedly 22–24 and 1–2 K/W for H 2 O and D 2 O, respectively. 60 The solution was then cooled and incubated at room temperature (~ 25°C) for 1 hour. For the laser experiments, a 15 µL aliquot of the solution was placed into a custom-made, hydrophilized sample cell consisting of a cover glass and a glass ring (Supplementary Fig. 7). After pouring the solution into the hand-made cell, it was sealed with another cover glass. The solution height was measured to be 160 ± 10 µm. Because prolonged incubation of L-Cys in the sample solutions (e.g., 1 day) can lead to the precipitation of "cystine" (Supplementary Fig. 8) due to cysteine oxidization, 61 solutions were always used within 5 hours of preparation. The sample cell was mounted on the microscope stage for subsequent laser experiments. Optical setup for CW laser trapping combined with Raman spectroscopic system. The optical setup for the combined CW laser trapping and Raman spectroscopic system is shown in Supplementary Fig. 9a. A near-infrared CW laser ( λ = 1064 nm) emitted from Nd 3+ :YVO 4 laser system (J201-BL-106C, Spectra-Physics) was employed for laser trapping. The laser beam was expanded and collimated using two convex lenses ( f 1 = 100 mm, f 2 = 200 mm) and then directed into a microscope (Eclipse Ti, Nikon). The laser beam was focused at the air/solution interface of the sample solution through an objective lens (60×, NA = 0.90, UPLFLN60X, Olympus). The laser power was adjusted using a half-wave plate and a polarizing beam splitter. The focal radius was estimated to be ~ 720 nm based on the Rayleigh criterion (0.61 × λ /NA), and this value was used to determine the laser intensity. The microscopic image was captured by a CCD camera through the objective lens. For Raman spectroscopy, a green CW laser ( λ = 532 nm, Millennia Pro s-Series 10 sJS, Spectra-Physics) served as the excitation source. Similar to the trapping laser, the excitation laser beam expanded and collimated using convex lenses ( f 1 = 100 mm, f 2 = 200 mm). The excitation laser was converted to circularly polarized light to minimize the influence of molecular alignment in crystal on the Raman scattering measurements. The scattered light was collected by a spectrometer (Shamrock, Andor) through an optical fiber (SR-OPT-8020, Andor) and detected by a cooled CCD camera (iDus, Andor). A notch filter positioned before the fiber blocked Rayleigh scattering and reflected light. Spectral analysis was performed using SOLIS software (Andor). Optical setup for HRR fs laser trapping and CW laser trapping The optical setup for HRR fs laser trapping and CW laser trapping is illustrated in Supplementary Fig. 9b. We utilized a mode-lock Ti:Sapphire laser system (Tsunami, Spectra-Physics) to generate high-repetition-rate femtosecond (HRR fs) laser pulses. This system was pumped by a green CW laser ( λ = 532 nm, Millennia-eV, Spectra-Physics) and produced pulses with a central wavelength of 800 nm, a pulse duration of 120 fs, and a repetition rate of 80 MHz. To ensure optimal beam quality, the laser pulses were expanded and collimated using a concave lens ( f 3 = -150 mm) and a convex lens ( f 4 = 250 mm). In addition to the HRR fs laser, we incorporated a near-infrared CW laser ( λ = 1064 nm, Nd 3+ : YVO 4 , MATRIX 1064-10-CW, Coherent) into the setup. This CW laser beam was also expanded and collimated, this time using two convex lens ( f 1 = 100 mm and f 2 = 200 mm). Both laser beams were then introduced to the inverted microscope (IX71, Olympus) and focused at the air/solution interface using the objective lens (60×, NA = 0.90). The focal radius of the HRR fs laser was estimated to be 540 nm. Finally, the polymorphs generated by laser irradiation were characterized using the optical system described in the previous section. Declarations Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Author contributions HT and TS conceptualized the research. HT carried out the experiments with guidance from HYY and TS. The first draft of manuscript was written by HT. All authors discussed the results and edited the manuscripts. Acknowledgements This work was partly supported by grants from the Japan Science and Technology Agency (JST) through the ACT-X program (No. JPMJAX23DC to HT), Japan Society for the Promotion of Science (JSPS) fellows (Nos. JP22KJ2214 and JP22J21666 to HT), and a KAKENHI Grant-in-Aid for Transformative Research Areas (A) "Revolution of Chiral Materials Science using Helical Light Fields" (No. JP22H05138 to TS). Additional support was provided by the JSPS KAKENHI (Nos: JP24H01138, JP23K18576, JP22H00302, JP22H05423, and JP19KK0128 to HYY, JP24KK0106 to HYY and HT), Amada Foundation, and Asahi Glass Foundation. 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Moggach SA, et al. High-pressure polymorphism in L-cysteine: the crystal structures of L-cysteine-III and L-cysteine-IV. Acta Crystallogr Sect B: Struct Sci 62, 296–309 (2006). Minkov VS, Goryainov SV, Boldyreva EV, Görbitz CH. Raman study of pressure-induced phase transitions in crystals of orthorhombic and monoclinic polymorphs of L‐cysteine: dynamics of the side chain. J Raman Spectrosc 41, 1748–1758 (2010). Parker SF. Assignment of the vibrational spectrum of l-cysteine. Chem Phys 424, 75–79 (2013). Liu S-F, Wang Y, Lin L. Laser-Induced Crystallization of Amino Acids through the Coordinated Effect of Optical Forces and Marangoni Convection. The Journal of Physical Chemistry A, (2024). Kruskal WH, Wallis WA. Use of Ranks in One-Criterion Variance Analysis. Journal of the American Statistical Association 47, 583–621 (1952). Han J, et al. Determination and Correlation of the Solubility of l-Cysteine in Several Pure and Binary Solvent Systems. J Chem Eng Data 65, 2649–2658 (2020). Yuyama K-i, Sugiyama T, Masuhara H. Millimeter-Scale Dense Liquid Droplet Formation and Crystallization in Glycine Solution Induced by Photon Pressure. J Phys Chem Lett 1, 1321–1325 (2010). Gowayed OY, Moosa T, Moratos AM, Hua T, Arnold S, Garetz BA. Dynamic Light Scattering Study of a Laser-Induced Phase-Separated Droplet of Aqueous Glycine. J Phys Chem B 125, 7828–7839 (2021). Chen T, Toyouchi S, Sugiyama T. Spectroscopic Analysis of Concentration Dynamics and Crystallization of Hen Egg-White Lysozyme under Optical Trapping Conditions. J Phys Chem C 127, 23340–23348 (2023). Markov IV. CRYSTAL GROWTH FOR BEGINNERS: Fundamentals of Nucleation, Crystal Growth and Epitaxy (World Scientific Publishing, 2003). Kitamura M. Polymorphism in adductive crystallization of Ni-complex in the presence of 1-methylnaphthalene. J Chem Eng Jpn 21, 589–595 (1988). Jiang Y, Narushima T, Okamoto H. Nonlinear optical effects in trapping nanoparticles with femtosecond pulses. Nature Physics 6, 1005–1009 (2010). Usman A, Chiang W-Y, Masuhara H. Optical trapping and polarization-controlled scattering of dielectric spherical nanoparticles by femtosecond laser pulses. J Photochem Photobio A 234, 83–90 (2012). Vogel A, Venugopalan V. Mechanisms of pulsed laser ablation of biological tissues. Chem Rev 103, 577–644 (2003). Vogel A, Noack J, Hüttman G, Paltauf G. Mechanisms of femtosecond laser nanosurgery of cells and tissues. Appl Phys B 81, 1015–1047 (2005). Soare A, et al. Crystal nucleation by laser-induced cavitation. Cryst Growth Des 11, 2311–2316 (2011). Nakamura K, Hosokawa Y, Masuhara H. Anthracene crystallization induced by single-shot femtosecond laser irradiation: Experimental evidence for the important role of bubbles. Cryst Growth Des 7, 885–889 (2007). Yoshikawa HY, et al. Laser energy dependence on femtosecond laser-induced nucleation of protein. Appl Phys A 93, 911–915 (2008). Yoshikawa HY, et al. Femtosecond laser-induced nucleation of protein in agarose gel. J Cryst Growth 311, 956–959 (2009). Iefuji N, et al. Laser-induced nucleation in protein crystallization: Local increase in protein concentration induced by femtosecond laser irradiation. J Cryst Growth 318, 741–744 (2011). Hidman N, Sardina G, Maggiolo D, Ström H, Sasic S. Numerical Frameworks for Laser-Induced Cavitation: Is Interface Supersaturation a Plausible Primary Nucleation Mechanism? Cryst Growth Des 20, 7276–7290 (2020). Takahashi H, Sugiyama T, Nakabayashi S, Yoshikawa HY. Crystallization from glacial acetic acid melt via laser ablation. Appl Phys Express 14, 045503 (2021). Tsuri Y, et al. Effects of pulse duration on laser-induced crystallization of urea from 300 to 1200 fs: impact of cavitation bubbles on crystal nucleation. Appl Phys A 128, 1–7 (2022). Nagalingam N, et al. Laser-Induced Cavitation for Controlling Crystallization from Solution. Phys Rev Lett 131, 124001 (2023). Takahashi H, et al. Spatiotemporal Control of Ice Crystallization in Supercooled Water via an Ultrashort Laser Impulse. J Phys Chem Lett 14, 4394–4402 (2023). Ito S, Sugiyama T, Toitani N, Katayama G, Miyasaka H. Application of Fluorescence Correlation Spectroscopy to the Measurement of Local Temperature in Solutions under Optical Trapping Condition. J Phys Chem B 111, 2365–2371 (2007). Ejgenberg M, Mastai Y. Biomimetic Crystallization of l-Cystine Hierarchical Structures. Cryst Growth Des 12, 4995–5001 (2012). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMovie1.mp4 Supplementary Movie 1 SupplementaryMovie2.mp4 Supplementary Movie 2 SupplementaryMovie3.mp4 Supplementary Movie 3 SupplementaryMovie4.mp4 Supplementary Movie 4 SupplementaryMovie5.mp4 Supplementary Movie 5 SupplementaryInformation.docx Supplementary Information Cite Share Download PDF Status: Published Journal Publication published 16 May, 2025 Read the published version in Communications Chemistry → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5777300","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":402079969,"identity":"87602ebd-3c2b-43cd-a223-c42239016ec6","order_by":0,"name":"Teruki Sugiyama","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYJCCAyCCH8phbCBai2QDXAszkVYZHCBWi/yM9IuHeWq2yRvf7jH8zMNgI7vhAP8xCbyG38gpOMxz7LbhtjtnjKV5GNKMNxxgZsOvRSIn4TAP223GbTdyzJh5GA4ngrTcwO8wkJZ/t+03zwBr+U9YC8ON9AOHedtuJ26QAGs5QFiLwZk3DAfn9t1OnnEjrVhyjkGy8czDzOY/8DqsPf3xhzffbtv2z0je+OFNhZ1s3/HGxwZ4HcbAY8DEA2ZwABWC1BKOSfYHjD+gDIJqR8EoGAWjYGQCAE6XUDHuyk2RAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9571-4388","institution":"National Yang Ming Chiao Tung University","correspondingAuthor":true,"prefix":"","firstName":"Teruki","middleName":"","lastName":"Sugiyama","suffix":""},{"id":402079970,"identity":"9ce4e438-4ed0-4d8b-910c-658e41bcc9b4","order_by":1,"name":"Hozumi Takahashi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hozumi","middleName":"","lastName":"Takahashi","suffix":""},{"id":402079971,"identity":"fdc95062-fe20-48d7-99e3-740082b3d4ef","order_by":2,"name":"Hiroshi Yoshikawa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Yoshikawa","suffix":""}],"badges":[],"createdAt":"2025-01-07 02:55:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5777300/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5777300/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42004-025-01554-7","type":"published","date":"2025-05-16T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73991547,"identity":"a3929823-10ae-4716-9db3-b9d5fedfeba4","added_by":"auto","created_at":"2025-01-16 17:15:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57213,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative crystallization behavior of L-Cys under CW laser trapping conditions. The laser intensity was set to 61 MW/cm\u003csup\u003e2\u003c/sup\u003e. The scale bar at 0 s represents 10 μm. A white spot shown at ~ 0 s is a focused 532 nm-laser used as an indicator of the air/solution interface.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/3be4a43f10c80bec89cbc9bc.png"},{"id":73990924,"identity":"acc145ae-eada-492f-a917-846fb92b0f93","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63204,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of CW laser intensity on L-cysteine crystallization probability and induction time. Linearly polarized light was used, and eight samples were tested at each laser intensity. Blue bars represent crystallization probability (%), defined as the percentage of trials resulting in crystal formation within 40 minutes of laser irradiation. Blue dots indicate the induction times for each laser intensity. The error bars represent the standard deviation.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/3e7414e19925e9fe340cc375.png"},{"id":73990919,"identity":"d2466d9c-3dc8-4077-81cb-173d6b773a16","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162250,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Representative temporal evolution of Raman intensity under various laser intensities. The dashed lines represent the linear fitting curves before and after crystal generation. Raman spectra were acquired every 5 seconds. The laser power of the excitation laser was set to ~0.1 W (24 MW/cm\u003csup\u003e2\u003c/sup\u003e). (b) Dependence of the concentration increase rate (left axis, turquoise blue) and concentration at the time of nucleation (right axis, purple) on laser intensity (\u003cem\u003en\u003c/em\u003e = 5). The marks are slightly offset along the horizontal axis to avoid overlapping.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/02a1714142e7819837e80d5d.png"},{"id":73990921,"identity":"5c649590-3bd4-4b80-b12e-9d826e151e91","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51583,"visible":true,"origin":"","legend":"\u003cp\u003eEvolution of L-Cys crystallization under HRR fs laser trapping. The laser intensity was maintained at 61 MW/cm\u003csup\u003e2\u003c/sup\u003e. Yellow arrows denote laser-induced bubbles, while green arrows highlight the emergence of stick-like crystals. Thin crystals are encircled in red. The scale bar at 0 s corresponds to 20 μm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/384daa637c6ee139866d4a40.png"},{"id":73991551,"identity":"2341ece7-39e8-4c9c-a7b8-bb9631ae0af9","added_by":"auto","created_at":"2025-01-16 17:15:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66517,"visible":true,"origin":"","legend":"\u003cp\u003eDependence of crystallization probability and bubble generation timing on laser intensity of HRR fs laser. Linearly polarized light was used as the trapping light source. The stable phase crystal was formed after ~ 10 s to ~ 10 minutes from bubble generation. Eight samples were tested in each condition. To properly evaluate induction time tendency, we assigned a value of 40 minutes to trials without crystal nucleation at 37 MW/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/eafe2a80cac2785e7c5450e5.png"},{"id":73990938,"identity":"33e8279c-557e-4aaa-a8be-8f5367573708","added_by":"auto","created_at":"2025-01-16 17:07:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":91675,"visible":true,"origin":"","legend":"\u003cp\u003ePolymorph control of L-Cys by sequential irradiation with CW and HRR fs lasers. The scale bars represent 20 μm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/82a5ddb1fb5f34df39e7893f.png"},{"id":73990930,"identity":"3e4fe259-11c0-457a-9594-f42411ba1327","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":126698,"visible":true,"origin":"","legend":"\u003cp\u003ePolymorph control of L-Cys crystals achieved by CW and HRR fs laser trapping.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/60a00bc8eb9ef7bc0765ef24.png"},{"id":82941840,"identity":"2651286a-ca78-40ca-8b76-157ba7f07edd","added_by":"auto","created_at":"2025-05-17 07:10:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1282873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/c292d480-d16b-4733-9fcc-686930036d9d.pdf"},{"id":73990923,"identity":"876a79de-3899-495f-b6d7-6c7d046f5db0","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5879929,"visible":true,"origin":"","legend":"Supplementary Movie 1","description":"","filename":"SupplementaryMovie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/bcd6cb2054d0a5c7814d5666.mp4"},{"id":73990934,"identity":"f2041786-67d8-4ef5-b173-3460251a43e8","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23363814,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie 2\u003c/p\u003e","description":"","filename":"SupplementaryMovie2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/77ef01936f78f6c62a566286.mp4"},{"id":73990935,"identity":"df412a50-7435-45e6-8281-f553719fa071","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":22953629,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie 3\u003c/p\u003e","description":"","filename":"SupplementaryMovie3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/65b177c0b4bed4048458f548.mp4"},{"id":73990955,"identity":"b60421e5-320f-47b2-97de-df01bde456cb","added_by":"auto","created_at":"2025-01-16 17:07:46","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":97986880,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie 4\u003c/p\u003e","description":"","filename":"SupplementaryMovie4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/6988ef6b7174b5c4b5b6f9b2.mp4"},{"id":73990933,"identity":"d539ce72-943b-4f60-a2ee-ea3f0ed29d46","added_by":"auto","created_at":"2025-01-16 17:07:43","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":8005980,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Movie 5\u003c/p\u003e","description":"","filename":"SupplementaryMovie5.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/89c5604947b40dddba743255.mp4"},{"id":73990951,"identity":"f3d9974d-938c-4ebb-8032-88355c1a4a62","added_by":"auto","created_at":"2025-01-16 17:07:44","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":7029675,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5777300/v1/8de126dc3a8211a03369b890.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Selective Manipulation of L-Cysteine Crystal Polymorphs Using Focused Laser Beams","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCrystal polymorphism \u0026mdash;the ability of a material to exist in more than one crystalline form\u0026mdash; has garnered significant attention across diverse scientific and industrial fields due to the distinct physical and chemical properties exhibited by different polymorphs. In the pharmaceutical sector, for instance, polymorphism profoundly influences critical drug characteristics such as solubility, stability, and bioavailability, all of which directly impact the drug's efficacy and safety.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Similarly, in materials science, polymorphism affects the mechanical, optical, and electronic properties of materials, influencing the performance of semiconductors, pigments, and energetic materials.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Consequently, developing effective strategies for the precise control of crystal polymorphs is crucial for designing crystalline materials with tailored properties. Traditionally, polymorphic control has been achieved by adjusting environmental parameters such as temperature, concentration, and solvent.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e However, controlling the polymorphism of organic compounds is challenging due to the relatively weaker intermolecular forces driving their crystallization, such as van der Waals and hydrogen bonding. Even with a systematic screening of environmental parameters, the precise and reproducible preparation of the desired number of crystals and crystal polymorphs in organic compounds often remains difficult.\u003c/p\u003e \u003cp\u003eRecent research has focused on the physical aspects of laser irradiation, such as electric field effects and heat generation, as a promising external perturbation for controlling the crystallization of various organic compounds.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Laser-based techniques such as Non-Photochemical Laser-Induced Nucleation (NPLIN) and laser ablation have shown potential in achieving polymorphic control, particularly in amino acids and pharmaceutical compounds.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Recent studies suggest that crystal nucleation and polymorphic selectivity through these methods are attributed to photothermally and/or photochemically produced bubbles, which locally increases solute concentrations and/or enhances heterogeneous nucleation.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eLaser trapping, a technique that allows for the contactless manipulation and condensation of substances at the nanometer- and micrometer-scales,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e has also emerged as a potential tool for the precise control of crystallization events.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Studies have demonstrated that focused irradiation with continuous-wave (CW) laser at the air/solution interface can induce crystal formation without bubble generation, and the polymorphs of the resulting crystals are highly dependent on laser parameters (laser intensity, polarization mode) as well as environmental conditions (e.g., initial concentration).\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e In terms of its mechanism, optical force, arising from the interaction between laser\u0026rsquo;s electric and solutes (theoretically treatment is summarized in Note 1), plays a crucial role in condensing the solutes at/around laser focus.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Interestingly, utilizing a high-repetition-rate femtosecond (HRR fs) laser instead of a CW laser can induce not only trapping effects but also bubble generation,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e a phenomenon often observed in studies of NPLIN and laser ablation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e This bubble generation contributes to polymorphic control in several compounds.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e While the condensation of solutes through trapping effects is likely essential for inducing crystal nucleation,\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e the critical factor governing polymorphic control remains elusive. Further investigation is necessary to achieve precise polymorphic manipulation.\u003c/p\u003e \u003cp\u003eIn this study, we investigated polymorph control in L-cysteine (L-Cys), an amino acid commonly used in pharmaceuticals and food additives,\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e by employing focused irradiation with CW and HRR fs lasers. L-Cys has also served as model compounds for polymorphic investigations.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Our findings revealed that CW laser trapping exclusively produced the \u003cem\u003eα\u003c/em\u003e-form (metastable phase), while HRR fs laser trapping consistently yielded the \u003cem\u003eβ\u003c/em\u003e-form (stable phase). Additionally, we introduced a novel approach for single crystal fabrication by strategically switching between CW and HRR fs laser irradiation during the crystallization process. This technique allows for precise manipulation of the polymorphic outcome and paves the way for the controlled growth of L-Cys single crystals with desired properties.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Continuous-Wave Laser Irradiation on L-Cysteine Crystallization\u003c/h2\u003e \u003cp\u003eA near-infrared CW laser with a wavelength of 1064 nm was focused at the air/solution interface of an unsaturated L-Cys/D\u003csub\u003e2\u003c/sub\u003eO solution with a supersaturation value of 0.84 (also see Methods). Here, the saturation value (\u003cem\u003eSS\u003c/em\u003e) was defined as the \u003cem\u003eC\u003c/em\u003e/\u003cem\u003eC\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e, where \u003cem\u003eC\u003c/em\u003e and \u003cem\u003eC\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e are the concentration of the sample and the saturated concentration (1.16 mol/kg at 25 \u0026ordm;C, Supplementary Note 2 and Supplementary Fig.\u0026nbsp;1), respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Movie S1 show the crystallization behavior of L-Cys under CW laser trapping. No discernible changes were observed immediately after laser irradiation (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0 s). After several tens of minutes, a thin, rod-like crystal was formed at the laser focus (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1165 s), subsequently developing into a hexagonal crystal with continued laser irradiation (\u003cem\u003et\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;1191 s). The polymorphs of the generated L-Cys crystals were characterized by Raman spectroscopy (cf. Supplementary Note 3 and Supplementary Fig.\u0026nbsp;2). Throughout the crystallization process (\u003cem\u003et\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;1165 s), Raman spectra confirmed the consistent presence of the \u003cem\u003eα\u003c/em\u003e-form. Previous studies of polymorphism control using laser trapping have indicated the significant influence of laser parameters such as intensity and polarization on crystal polymorphs, crystallization probability, and induction time.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Therefore, we investigated the dependence of these parameters on the intensity of the linearly polarized CW laser. In this study, crystallization probability was defined as the ratio of successful trials, where crystal formation was observed within 40 minutes of laser irradiation, to the total number of trials. For example, if crystal formation was observed for 8 samples out of 10 trials, the crystallization probability would be 80%. To properly evaluate the tendency of the induction time, if crystallization was not observed within 40 minutes, the induction time was recorded as 40 minutes, indicating that the actual induction time is equal to or greater than 40 minutes. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the results. Reliable L-Cys crystallization was observed at a laser intensity of 61 MW/cm\u003csup\u003e2\u003c/sup\u003e and higher, while crystallization was inconsistent at 37 MW/cm\u003csup\u003e2\u003c/sup\u003e. The induction time exhibited a decreasing trend with increasing laser intensity. This observation regarding the induction time was statically validated through the Kruskal-Wallis test,\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e which confirmed the significance of the difference (\u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0049%). Notably, despite variations in both crystallization probability and induction time, L-Cys crystals generated via CW laser trapping consistently exhibited the \u003cem\u003eα\u003c/em\u003e-form. This exclusive formation of the \u003cem\u003eα\u003c/em\u003e-form persisted across various conditions, including different polarization modes at 61 MW/cm\u003csup\u003e2\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;3) and a systematic screening of solution concentrations from 0.98 to 1.35 mol/kg (\u003cem\u003eSS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.98\u0026ndash;1.16), all of which consistently resulted in \u003cem\u003eα\u003c/em\u003e-form crystallization.\u003c/p\u003e \u003cp\u003eIt is widely known that supersaturation plays a critical role in determining the polymorphic outcome of crystallization. As supersaturation is governed by both temperature and concentration, it is crucial to estimate the increase in both under CW laser trapping conditions to elucidate the mechanism underlying the exclusive formation of the metastable \u003cem\u003eα\u003c/em\u003e-form. To observe the temporal evolution of concentration under laser trapping, we employed Raman spectroscopy to monitor the concentration dynamics of L-Cys. The L-Cys/D\u003csub\u003e2\u003c/sub\u003eO solution exhibits several peaks between 200 and 2000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;4a), and the intensity of some peaks (e.g., ~\u0026thinsp;690, ~1430, and ~\u0026thinsp;1870 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was confirmed to be proportional to the L-Cys concentration. We tracked the concentration using the highest peak at ~\u0026thinsp;1870 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (calibration curve: Supplementary Fig.\u0026nbsp;4b), which allowed for rapid measurement of Raman intensity (5 seconds per spectrum). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows the representative temporal evolution of Raman intensity at different laser intensities (37, 61, and 86 MW/cm\u003csup\u003e2\u003c/sup\u003e). The intensity initially increased linearly with irradiation time, irrespective of the laser intensity. The intensity suddenly jumped upon crystallization, possibly facilitating the estimation of nucleation timing. The slope of the linear fitting curve before crystallization was (0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12) \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e a.u./s at 37 MW/cm\u003csup\u003e2\u003c/sup\u003e, (1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18) \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e a.u./s at 61 MW/cm\u003csup\u003e2\u003c/sup\u003e, and (2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16) \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e a.u./s at 86 MW/cm\u003csup\u003e2\u003c/sup\u003e. From the calibration curve (Supplementary Fig.\u0026nbsp;4b), the concentration increase rates were 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mmol/kg/s at 37 MW/cm\u003csup\u003e2\u003c/sup\u003e, 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mmol/kg/s at 61 MW/cm\u003csup\u003e2\u003c/sup\u003e, and 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mmol/kg/s at 86 MW/cm\u003csup\u003e2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, left axis), with significant differences confirmed by the Kruskal-Wallis test (\u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.19%). These differences explain the observed shorter induction time at higher laser intensities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Unexpectedly, despite the varying rates of concentration increase, the actual concentration of L-Cys at the point of crystallization, as estimated from the Raman intensity at the intersection of the linear fitting curves before and after crystallization, was consistently around 2.5 mol/kg for all laser intensities, with no significant differences (\u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;57%). This remarkable consistency reveals a critical threshold for supersaturation that must be attained to initiate the formation of the \u003cem\u003eα\u003c/em\u003e-form, regardless of how quickly that supersaturation level is reached.\u003c/p\u003e \u003cp\u003eNext, to accurately assess the supersaturation and its impact on the polymorphic outcome, we need to consider the effect of temperature elevation on L-Cys solubility. It is also essential to estimate the local temperature increase caused by laser irradiation, particularly since, for L-Cys, temperature influences which polymorph is formed (transition temperature: \u0026ge; 32\u0026deg;C, cf. Supplementary Note 3). Therefore, we investigated the temperature elevation of the L-Cys/D\u003csub\u003e2\u003c/sub\u003eO solution upon CW laser irradiation. Based on the spectroscopic analysis (Supplementary Note 4 and Supplementary Fig.\u0026nbsp;5), the temperature elevation coefficient at the laser focus was determined to be 1.3\u0026deg;C/W. Consequently, in the laser intensity regime in this study, the maximum temperature elevation was estimated to be ~\u0026thinsp;2\u0026deg;C (25\u0026deg;C \u0026rarr; 27\u0026deg;C). Since this achieved temperature is considerably lower than the transition temperature (\u0026ge;\u0026thinsp;32\u0026deg;C), we concluded that temperature elevation from laser irradiation should not contribute to the exclusive crystallization of the \u003cem\u003eα\u003c/em\u003e-form. Hence, we focused on calculating the supersaturation at the point of crystallization. According to a previous study,\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e a temperature elevation from 25\u0026deg;C to 27\u0026deg;C leads to a 4% increase in L-Cys solubility in H\u003csub\u003e2\u003c/sub\u003eO (see Supplementary Note 5). Assuming that the solubility in D\u003csub\u003e2\u003c/sub\u003eO also increases by 4%, the supersaturation at crystallization was estimated to be 2.5/(1.16 \u0026times; 1.04)\u0026thinsp;=\u0026thinsp;2.1. This value is significantly higher than that required for spontaneous crystallization (\u003cem\u003eSS\u003c/em\u003e\u0026thinsp;~\u0026thinsp;1.2), indicating that laser trapping induces a highly supersaturated state prior to nucleation.\u003c/p\u003e \u003cp\u003eIt may seem surprising that laser trapping can induce such a high degree of supersaturation. Still, this phenomenon has been observed in previous studies on other amino acid and protein solutions.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e It is important to note that nucleation frequency is governed by the magnitude of the activation energy required for nucleation, which is inversely proportional to the square of the difference in chemical potential between the solution and crystal and proportional to the cube of the surface free energy of the generating crystal.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Additionally, the surface free energy of the metastable phase is generally smaller than that of the stable phase due to the relationship between the enthalpy of the metastable phase and the stable phase. Therefore, under conditions where supersaturation is sufficiently high for both polymorphs, the activation energy required for nucleation of the metastable phase is smaller than that of the stable phase, making the metastable phase kinetically accessible. This could explain the exclusive crystallization of the metastable \u003cem\u003eα\u003c/em\u003e-form upon CW laser trapping.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of high repetition-rate femtosecond laser on L-Cys crystallization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the preceding section, we observed that CW laser irradiation consistently resulted in the crystallization of the \u003cem\u003eα\u003c/em\u003e-form of L-Cys despite adjusting parameters such as laser intensity and solution concentration. This observation led us to hypothesize that alternative laser-based perturbations might be required to induce the crystallization of the \u003cem\u003eβ\u003c/em\u003e-form. To explore this possibility, we employed a focused HRR fs laser, which not only induces laser trapping but also causes additional phenomena such as laser ablation.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Movie S2 show the representative crystallization behavior of L-Cys under HRR fs laser trapping. This experiment also employed the unsaturated L-Cys/D\u003csub\u003e2\u003c/sub\u003eO solution with \u003cem\u003eSS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.84. No apparent change was observed immediately after the laser irradiation. After several minutes of irradiation, bubbles were suddenly generated at the laser focus (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1665 s). During bubble generation, small crystals (indicated by a red circle in the figure) were repeatedly produced and dissolved around the laser focus. Suddenly, rectangular crystals (indicated by green arrows in the figure) were yielded around the laser focus (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1675 s). These rectangular crystals grew substantially with continued laser irradiation. When the growing crystals were trapped at the laser focus, they underwent ablation, resulting in poly-crystallization (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1680 s). Surprisingly, Raman spectroscopy confirmed that these rectangle crystals correspond to the stable phase of \u003cem\u003eβ\u003c/em\u003e-form, which was never observed with CW laser trapping. Due to their limited size, we were unable to characterize the small crystals that formed prior to β-form crystallization using Raman spectroscopy. Nevertheless, we employed alternative methods to determine that these crystals were likely the \u003cem\u003eα\u003c/em\u003e-form, as detailed in a later section.\u003c/p\u003e \u003cp\u003eTo examine the impact of HRR fs laser irradiation on the crystallization behavior of L-Cys, we investigated the dependence of eventual polymorphism, crystallization probability, and bubble generation timing on the intensity of the linearly polarized laser. The results are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Crystals always appeared at 61 MW/cm\u003csup\u003e2\u003c/sup\u003e and 86 MW/cm\u003csup\u003e2\u003c/sup\u003e, while some trials failed at 37 MW/cm\u003csup\u003e2\u003c/sup\u003e. The bubble generation timing was significantly accelerated with increasing laser intensity (\u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.08%). While such crystallization behavior is similar to that of the CW laser (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the HRR fs laser irradiation always produced the stable phase of \u003cem\u003eβ\u003c/em\u003e-form eventually. This result underscores that not only laser trapping but also additional phenomena characteristic of HRR fs laser contributed to the generation of \u003cem\u003eβ\u003c/em\u003e-form.\u003c/p\u003e \u003cp\u003eWe now discuss the crystallization and polymorphism mechanism of L-Cys by HRR fs laser irradiation. Unlike the case of CW laser irradiation, successive irradiation with HRR fs laser suddenly produces bubbles at the laser focus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, t\u0026thinsp;=\u0026thinsp;1665 s). Such bubble generation has also been observed in the experiments on HRR fs laser trapping-induced crystallization using other amino acid solutions.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e This phenomenon can be explained as follows: the focused irradiation with a HRR fs laser induces a concentration increase at the laser focus due to its laser trapping effect.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e As the concentration is increased, the multiphoton absorption by solutes is enhanced due to the considerably higher peak intensity of the HRR fs laser (\u0026ge;\u0026thinsp;3.8 TW/cm\u003csup\u003e2\u003c/sup\u003e) compared to the CW laser (\u0026le;\u0026thinsp;86 MW/cm\u003csup\u003e2\u003c/sup\u003e). When the concentration reaches a certain level, the multiphoton absorption triggers laser ablation of the solution, producing bubbles.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e These generated bubbles may correspond to cavitation bubbles that are often produced by irradiation with ultrashort laser pulses.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Indeed, it has been reported that bubble generation by laser ablation of solutions can induce crystal nucleation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e As for its mechanism, several studies have proposed that local concentration increase and/or heterogeneous nucleation at the bubble surface plays an important role in crystal nucleation by laser ablation.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e The preferential nucleation of the α-form polymorph can be attributed to the localized increase in solute concentration induced by laser trapping and the subsequent generation of cavitation bubbles. This localized concentration increase leads to a high degree of supersaturation, favoring the nucleation of the α-form, which is characterized by lower surface free energy. As the \u003cem\u003eα\u003c/em\u003e-form crystals grow, solute molecules are consumed, leading to a decrease in the supersaturation level within the vicinity of the laser focus. The decrease in supersaturation causes a shift in the dominant factor for nucleation, from surface free energy to chemical potential. Consequently, subsequent bubble generation events within this region of reduced supersaturation promote the nucleation of the \u003cem\u003eβ\u003c/em\u003e-form polymorph, which possesses a lower chemical potential and becomes thermodynamically favored under these conditions. This phenomenon exemplifies the delicate interplay between supersaturation, surface free energy, and chemical potential in determining the polymorphic outcome of crystallization processes. The initial dominance of surface free energy under high supersaturation conditions gives way to the dominance of chemical potential as supersaturation decreases, effectively dictating the transition from α-form to \u003cem\u003eβ\u003c/em\u003e-form nucleation. In fact, such a conversion from metastable polymorph to stable polymorph has also been observed in HRR fs laser trapping of L-phenylalanine.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSelective Manipulation of Polymorphs by Combination of CW Laser and HRR Fs Laser\u003c/h3\u003e\n\u003cp\u003eThe results presented in the preceding sections demonstrate that the eventual polymorph of L-Cys can be controlled by selecting the appropriate laser irradiation mode: CW laser irradiation consistently yields the \u003cem\u003eα\u003c/em\u003e-form, while HRR fs laser irradiation ultimately produces the \u003cem\u003eβ\u003c/em\u003e-form. Building upon these findings, we explored the possibility of achieving dynamic control over the eventual number of the crystals and the resulting polymorph by strategically combining CW and HRR fs laser irradiation.\u003c/p\u003e \u003cp\u003eFor this purpose, we designed an experiment where we switched between CW and HRR fs laser irradiation during crystal growth. As shown in the leftmost image in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, we first irradiated the HRR fs laser at 86 MW/cm\u003csup\u003e2\u003c/sup\u003e into the L-Cys solution until bubble generation was observed at the laser focus. Immediately confirming the bubble generation, we switched to CW laser irradiation. This change in irradiation mode led to the continued growth of the large single \u003cem\u003eα\u003c/em\u003e-form crystal (~\u0026thinsp;200 \u0026micro;m) without producing the \u003cem\u003eβ\u003c/em\u003e-form (the upper path in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplementary Movie 3). This result indicates that switching to CW laser irradiation before \u003cem\u003eβ\u003c/em\u003e-form crystallization by the subsequent HRR fs laser irradiation can promote the growth of \u003cem\u003eα\u003c/em\u003e-form crystals. This result also emphasizes that the small crystals observed before \u003cem\u003eβ\u003c/em\u003e-form crystallization are more likely to the \u003cem\u003eα\u003c/em\u003e-form crystals. Furthermore, we confirmed that re-switching to HRR fs laser irradiation after sufficient growth of the \u003cem\u003eα\u003c/em\u003e-form crystal eventually still led to the formation of the \u003cem\u003eβ\u003c/em\u003e-form (Supplementary Fig.\u0026nbsp;6 and Supplementary Movie 4). Specifically, re-switching to HRR fs laser initially induced etching of the crystals surface with generating bubbles. Approximately 2 minutes after re-switching to HRR fs laser, the \u003cem\u003eβ\u003c/em\u003e-form crystals suddenly appeared around the laser focus. This observation underscores the role of HRR fs laser irradiation in inducing \u003cem\u003eβ\u003c/em\u003e-form crystallization.\u003c/p\u003e \u003cp\u003eThe combination of CW laser and HRR fs laser also offers another advantage. Activating CW laser immediately after the crystallization of the \u003cem\u003eβ\u003c/em\u003e-form resulted in single crystal growth without poly-crystallization (the bottom path in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Supplementary Movie 5). This precise control over single crystal growth is challenging to achieve with the HRR fs laser alone, as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Thus, precise spatial and temporal manipulation of CW and HRR fs laser irradiation enables the preparation of single L-Cys crystals with the desired polymorph. This technique offers a new level of control over the crystallization and polymorphism process, allowing for the targeted production of specific polymorphs.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study has delved into the selective manipulation of L-cysteine polymorphism, revealing the remarkable ability of focused laser irradiation to guide the polymorphism selectively. We have demonstrated that continuous-wave (CW) laser irradiation consistently yields the metastable \u003cem\u003eα\u003c/em\u003e-form, while high repetition-rate femtosecond (HRR fs) laser irradiation favors the stable \u003cem\u003eβ\u003c/em\u003e-form. This intriguing divergence stems from the distinct mechanisms triggered by each laser type. CW laser irradiation generates a highly supersaturated state, promoting the rapid formation of the kinetically accessible \u003cem\u003eα\u003c/em\u003e-form. Conversely, HRR fs laser irradiation induces bubble generation and localized concentration increases, ultimately tipping the scales toward the thermodynamically stable \u003cem\u003eβ\u003c/em\u003e-form.\u003c/p\u003e \u003cp\u003eTaking this a step further, we achieved dynamic control over the crystallization pathway by strategically alternating between CW and HRR fs laser irradiation. This innovative approach, visually summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, allowed us to achieve remarkable results in crystal engineering, such as growing large single crystals of the \u003cem\u003eα\u003c/em\u003e-form and preventing polycrystallization of the \u003cem\u003eβ\u003c/em\u003e-form. The implications of this work extend far beyond L-cysteine. By harnessing the precise interplay of laser parameters and material responses, we can envision a future where we can manipulate crystal structures with unprecedented accuracy. This promises to revolutionize fields ranging from pharmaceuticals and electronics to optics and catalysis. Our findings illuminate a path towards a new era of materials design, where light serves as a sculptor of matter at the molecular level.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eSample preparation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eL-cysteine (L-Cys, \u0026ge; 99%, Sigma-Aldrich) was used as received without further purification. L-Cys crystals were completely dissolved in deuterium oxide (D\u003csub\u003e2\u003c/sub\u003eO) by stirring at 70\u0026deg;C for 15 minutes. D\u003csub\u003e2\u003c/sub\u003eO was chosen as the solvent instead of H\u003csub\u003e2\u003c/sub\u003eO to minimize temperature increases caused by the near-infrared CW laser irradiation. The temperature elevation coefficients (thermal absorption coefficients) at 1064 nm are reportedly 22\u0026ndash;24 and 1\u0026ndash;2 K/W for H\u003csub\u003e2\u003c/sub\u003eO and D\u003csub\u003e2\u003c/sub\u003eO, respectively.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e The solution was then cooled and incubated at room temperature (~\u0026thinsp;25\u0026deg;C) for 1 hour. For the laser experiments, a 15 \u0026micro;L aliquot of the solution was placed into a custom-made, hydrophilized sample cell consisting of a cover glass and a glass ring (Supplementary Fig.\u0026nbsp;7). After pouring the solution into the hand-made cell, it was sealed with another cover glass. The solution height was measured to be 160\u0026thinsp;\u0026plusmn;\u0026thinsp;10 \u0026micro;m. Because prolonged incubation of L-Cys in the sample solutions (e.g., 1 day) can lead to the precipitation of \"cystine\" (Supplementary Fig.\u0026nbsp;8) due to cysteine oxidization,\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e solutions were always used within 5 hours of preparation. The sample cell was mounted on the microscope stage for subsequent laser experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOptical setup for CW laser trapping combined with Raman spectroscopic system.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe optical setup for the combined CW laser trapping and Raman spectroscopic system is shown in Supplementary Fig.\u0026nbsp;9a. A near-infrared CW laser (\u003cem\u003eλ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1064 nm) emitted from Nd\u003csup\u003e3+\u003c/sup\u003e:YVO\u003csub\u003e4\u003c/sub\u003e laser system (J201-BL-106C, Spectra-Physics) was employed for laser trapping. The laser beam was expanded and collimated using two convex lenses (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;100 mm, \u003cem\u003ef\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;200 mm) and then directed into a microscope (Eclipse Ti, Nikon). The laser beam was focused at the air/solution interface of the sample solution through an objective lens (60\u0026times;, NA\u0026thinsp;=\u0026thinsp;0.90, UPLFLN60X, Olympus). The laser power was adjusted using a half-wave plate and a polarizing beam splitter. The focal radius was estimated to be ~\u0026thinsp;720 nm based on the Rayleigh criterion (0.61\u0026thinsp;\u0026times;\u0026thinsp;\u003cem\u003eλ\u003c/em\u003e/NA), and this value was used to determine the laser intensity. The microscopic image was captured by a CCD camera through the objective lens.\u003c/p\u003e \u003cp\u003eFor Raman spectroscopy, a green CW laser (\u003cem\u003eλ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;532 nm, Millennia Pro s-Series 10 sJS, Spectra-Physics) served as the excitation source. Similar to the trapping laser, the excitation laser beam expanded and collimated using convex lenses (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;100 mm, \u003cem\u003ef\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;200 mm). The excitation laser was converted to circularly polarized light to minimize the influence of molecular alignment in crystal on the Raman scattering measurements. The scattered light was collected by a spectrometer (Shamrock, Andor) through an optical fiber (SR-OPT-8020, Andor) and detected by a cooled CCD camera (iDus, Andor). A notch filter positioned before the fiber blocked Rayleigh scattering and reflected light. Spectral analysis was performed using SOLIS software (Andor).\u003c/p\u003e\n\u003ch3\u003eOptical setup for HRR fs laser trapping and CW laser trapping\u003c/h3\u003e\n\u003cp\u003eThe optical setup for HRR fs laser trapping and CW laser trapping is illustrated in Supplementary Fig.\u0026nbsp;9b. We utilized a mode-lock Ti:Sapphire laser system (Tsunami, Spectra-Physics) to generate high-repetition-rate femtosecond (HRR fs) laser pulses. This system was pumped by a green CW laser (\u003cem\u003eλ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;532 nm, Millennia-eV, Spectra-Physics) and produced pulses with a central wavelength of 800 nm, a pulse duration of 120 fs, and a repetition rate of 80 MHz. To ensure optimal beam quality, the laser pulses were expanded and collimated using a concave lens (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e = -150 mm) and a convex lens (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;250 mm). In addition to the HRR fs laser, we incorporated a near-infrared CW laser (\u003cem\u003eλ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1064 nm, Nd\u003csup\u003e3+\u003c/sup\u003e: YVO\u003csub\u003e4\u003c/sub\u003e, MATRIX 1064-10-CW, Coherent) into the setup. This CW laser beam was also expanded and collimated, this time using two convex lens (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;100 mm and \u003cem\u003ef\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;200 mm). Both laser beams were then introduced to the inverted microscope (IX71, Olympus) and focused at the air/solution interface using the objective lens (60\u0026times;, NA\u0026thinsp;=\u0026thinsp;0.90). The focal radius of the HRR fs laser was estimated to be 540 nm. Finally, the polymorphs generated by laser irradiation were characterized using the optical system described in the previous section.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003ch3\u003eData Availability\u003c/h3\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eHT and TS conceptualized the research. HT carried out the experiments with guidance from HYY and TS. The first draft of manuscript was written by HT. All authors discussed the results and edited the manuscripts.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was partly supported by grants from the Japan Science and Technology Agency (JST) through the ACT-X program (No. JPMJAX23DC to HT), Japan Society for the Promotion of Science (JSPS) fellows (Nos. JP22KJ2214 and JP22J21666 to HT), and a KAKENHI Grant-in-Aid for Transformative Research Areas (A) \"Revolution of Chiral Materials Science using Helical Light Fields\" (No. JP22H05138 to TS). Additional support was provided by the JSPS KAKENHI (Nos: JP24H01138, JP23K18576, JP22H00302, JP22H05423, and JP19KK0128 to HYY, JP24KK0106 to HYY and HT), Amada Foundation, and Asahi Glass Foundation. We also acknowledge the support from the National Science and Technology Council (NSTC) of Taiwan (MOST 113-2113-M-A49-023 to TS) and the Center for Emergent Functional Matter Science of National Yang Ming Chiao Tung University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) of Taiwan (NSTC 111-2634-F-A49-007 to TS). Our gratitude also extends to the support from the Japan-Taiwan Exchange Association.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrittain HG. Polymorphism in pharmaceutical solids. Drugs and the pharmaceutical sciences 95, 183\u0026ndash;226 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorissette SL, \u003cem\u003eet al.\u003c/em\u003e High-throughput crystallization: polymorphs, salts, co-crystals and solvates of pharmaceutical solids. 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Application of Fluorescence Correlation Spectroscopy to the Measurement of Local Temperature in Solutions under Optical Trapping Condition. J Phys Chem B 111, 2365\u0026ndash;2371 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEjgenberg M, Mastai Y. Biomimetic Crystallization of l-Cystine Hierarchical Structures. Cryst Growth Des 12, 4995\u0026ndash;5001 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5777300/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5777300/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe selective manipulation of crystal polymorphism holds profound implications across diverse scientific and industrial fields, as distinct polymorphs exhibit unique physical and chemical properties. This study demonstrates selective polymorphic manipulation by laser trapping \u0026ndash; a technique enabling contactless manipulation and condensation of matter at the nanometer-scale and micrometer-scale. L-cysteine, a ubiquitous amino acid employed in pharmaceuticals and food additives, was targeted. We reveal that continuous-wave laser irradiation yields single crystals of the metastable polymorph, whereas successive irradiation with high-repetition-rate femtosecond laser pulses induces poly-crystallization of the stable form. Crucially, by strategically alternating between these two laser modalities during crystal growth, we can open up new crystallization pathways, including the generation of single crystals of the stable phase. These findings underscore the significant potential of focused laser beams for precision polymorphic engineering, paving the way for the development of advanced materials with tailored properties.\u003c/p\u003e","manuscriptTitle":"Selective Manipulation of L-Cysteine Crystal Polymorphs Using Focused Laser Beams","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-16 17:07:38","doi":"10.21203/rs.3.rs-5777300/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d5a21269-73fd-4542-9291-cf74712fc87e","owner":[],"postedDate":"January 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42855128,"name":"Physical sciences/Chemistry/Physical chemistry/Chemical physics"},{"id":42855129,"name":"Physical sciences/Materials science/Condensed-matter physics/Structure of solids and liquids"}],"tags":[],"updatedAt":"2025-05-17T07:10:25+00:00","versionOfRecord":{"articleIdentity":"rs-5777300","link":"https://doi.org/10.1038/s42004-025-01554-7","journal":{"identity":"communications-chemistry","isVorOnly":false,"title":"Communications Chemistry"},"publishedOn":"2025-05-16 04:00:00","publishedOnDateReadable":"May 16th, 2025"},"versionCreatedAt":"2025-01-16 17:07:38","video":"","vorDoi":"10.1038/s42004-025-01554-7","vorDoiUrl":"https://doi.org/10.1038/s42004-025-01554-7","workflowStages":[]},"version":"v1","identity":"rs-5777300","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5777300","identity":"rs-5777300","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}