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The effect of gel–solutioninterfaces on the femtosecondlaser-induced nucleation of proteins was investigated. Using hen eggwhite lysozyme, we found that the nucleation efficiency could be modulatedby the distance between the laser focus and agarose gel surfaces (h) and the gel concentration (c). In particular,laser irradiation near the soft gel surface (h=50 μm, c= 0.5%) could induce nucleation atvery low supersaturation, where no nucleation could be induced inthe bulk solutions even with femtosecond laser irradiation. Such enhancementof the nucleation was also confirmed for the membrane protein, acriflavineresistance protein B (AcrB), and an organic compound, N-(4-hydroxyphenyl)acetamide(paracetamol). To gain further insights into the role of gel–solutioninterfaces, we conducted fast imaging of cavitation bubbles, whichare known to locally concentrate supersaturated solutions and thusact as a trigger for laser-induced nucleation. We found that the cavitationbubbles shrank asymmetrically near the soft gel surfaces and thenfinally collapsed toward the solution side. This is in contrast tothe symmetric collapse in bulk solutions, which generates a largeamount heat and chemical decomposition at the focus. In addition,cavitation bubbles generated near stiff gel surfaces (c≥ 2%) caused an axial liquid jet toward the gel surfaces,which would disturb the formation of locally concentrated regions.These results suggest that gel–solution interfaces significantlymodulate cavitation bubble dynamics and can be cues to achieve effectivenucleation. [ABSTRACT FROM AUTHOR]

We developed a spatially precise, soft microseeding methodforthe production of single protein crystals that are suitable for X-raycrystallographic studies. We used focused femtosecond laser pulsesto produce, via multiphoton absorption processes, seed crystals fromsmall regions (∼1 μm2) of crystals. Hen egg-whitelysozyme seed crystals, produced in this manner, grew to be singlecrystals without any deterioration in their crystallinity. We alsovalidated the technique using polycrystals for the membrane protein,acriflavine resistance protein B, for which single crystals are verydifficult to obtain. In addition, we found that the shape of a tetragonallysozyme crystal prepared from the seed could be controlled by alteringthe time interval between the initiation of crystallization and laserablation. We also tried to comprehend the mechanism of femtosecondlaser-induced microseeding. We visualized the ablated surfaces ofthe lysozyme crystals by atomic force microscopy and by laser confocalmicroscopy combined with differential interference microscopy. Theresults obtained in this study clearly demonstrate that femtosecondlaser ablation of protein crystals is based on a photomechanical process,which ejects crystal fragments with little thermal damage. Femtosecondlaser ablation is indeed very promising to produce high quality proteinseed crystals from polycrystals or cracked crystals that are not suitablefor X-ray diffraction studies. [ABSTRACT FROM AUTHOR]

Growth of urea crystals in a supersaturated solution of urea was spatially controlled by irradiation with a femtosecond laser pulse. When a needlelike urea crystal was irradiated by a single femtosecond laser pulse with wavelength of 800 or 1470 nm through an objective lens, a new needlelike crystal grew from the laser focal spot (approximately micrometer sized). The number of the laser-induced subsequent crystals increased with laser energy. However, such crystal growth could not be induced by 1480 nm continuous wave (CW) laser irradiation. By application of the new phenomenon, patterning of the crystals was successfully demonstrated. Finally, the mechanism of the femtosecond laser-induced crystallization was discussed in terms of the crystal fragmentation caused by photomechanical effect. Growth of urea crystals in a supersaturated solution of urea was spatially controlled by irradiation with a femtosecond laser pulse. When a needlelike urea crystal was irradiated by a single femtosecond laser pulse with wavelength of 800 or 1470 nm, a new needlelike crystal grew from the laser focal spot. [ABSTRACT FROM AUTHOR]