标题: 雷射捕陷诱发溶菌酶结晶化机制的萤光显微光谱研究
Fluorescence microspectroscopic study of lysozyme crystallization mechanism under laser trapping
作者: 林咏伦
增原宏
Lin, Yung-Lun
Hiroshi, Masuhara
应用化学系硕博士班
关键字: 雷射捕陷;溶菌酶结晶化;萤光显微光谱;Fluorescence microspectroscopic study;Lysozyme crystallization;laser trapping
公开日期: 2016
摘要: 迄今,我们团队已经利用雷射捕陷技术成功地控制溶菌酶在过饱和重水溶液中的成核作用以及其结晶的成长。在雷射捕陷的作用下,溶菌酶团簇与分子于聚焦点所产生的高浓度溶菌酶丛集在此过程中扮演了非常重要的角色,溶菌酶的成核作用以及结晶的成长皆发生于这高浓度的丛集中。也就是说,对于这个高浓度溶菌酶丛集形成的动力学和微观的结构的瞭解是很重要且不可或缺的。在这项研究中,我们于溶菌酶溶液中加入萤光探针,并以萤光量测系统观测其在雷射捕陷下的动态与机制。
若丹明B作为萤光探针与溶菌酶重水缓冲溶液混合,将一连续波长之近红外光雷射聚焦于高于溶液的玻璃/溶液界面3微米的位置。此外,另一道400奈米的飞秒雷射作为萤光的激发光源引入到显微镜中,并分别聚焦在与近红外光雷射的焦点横向距离为10或20微米的位置上。萤光讯号被连结到时间相关单光子计数系统的单光子雪崩检光二极体所侦测,我们量测此染料掺杂溶液在雷射捕陷状态下,光子数及萤光衰减的变化。
在近红外雷射焦点外10和20微米的萤光强度随着雷射捕陷的时间逐渐增加;相反地,当捕陷光源被关掉后,萤光强度则开始下降。我们合理的认为萤光强度的变化是归因于大尺寸高浓度溶菌酶丛集的形成与消散。在雷射捕陷的状态下,萤光生命周期相较于初始溶液来的短;而在关掉捕陷光源后,萤光生命周期逐渐回升且较初始溶液来的长。前者是以因雷射捕陷作用下,可以以于聚焦焦点所产生的温度梯度分布的观点来解释;而后者则被认为是由于高浓度溶菌酶丛集内结构的变化,这也是结晶往往被限制在焦点周围附近毫米尺度范围空间内的原因。此外,我们也依据萤光生命周期实验所得到的结果来探讨所形成的丛集中类液相溶菌酶团簇的微观结构。我们相信,在探讨雷射捕陷作用下的成核作用以及大尺寸丛集形成的学说上,我们的微观研究创立了一个重要的里程碑。
We have so far demonstrated the control of nucleation and crystal growth of hen egg-white lysozyme (HEWL) by laser trapping at a glass/solution interfacial layer in the supersaturated D2O solution. One of important findings in those studies is that firstly laser trapping forms a large-sized highly-concentrated domain of HEWL liquid-like clusters and then crystal nucleation and crystal growth take place in the dense cluster domain. Namely, it is important and indispensable to understand the formation dynamics and microscopic structure of the dense domain for developing the laser trapping-induced nucleation and crystal growth. In this research, we studied the formation dynamics of the cluster domain by measuring fluorescence of a probe dye molecule added in the HEWL D2O solution under laser trapping conditions.
RhB was used as a probe molecule and mixed with a HEWL D2O buffer solution. A continuous-wave near-infrared (NIR) laser beam was focused at a position 3 μm above a glass/solution interface of the solution. A 400-nm femtosecond laser was also introduced to the microscope as a fluorescence excitation light source and focused at a point 10 or 20 μm laterally away from the focal spot of the NIR laser. Fluorescence was detected with a single photon counting avalanche photodiode which was connected to a time-correlated single photon counting system. We measured the photon count and the fluorescence decay under laser trapping conditions.
The fluorescence intensity measured at points 10 and 20 μm away from the focus of the NIR laser was gradually increased during the laser trapping. After the trapping laser was switched off, the fluorescence intensity was decreased. We considered that the florescence intensity increase and decrease are ascribed to the formation and dissolution of a large-sized highly-concentrated cluster domain. The fluorescence lifetime showed tendency to become shorter during laser trapping, whereas it was gradually increased after turning off the trapping laser. The former was explained from the viewpoint of temperature distribution generated from the focal spot of the trapping laser. The latter was considered be due to association structure change of the dense cluster domain, which results in the spatial confinement of the crystallization in a millimeter-scale around the focal spot. Furthermore, we discussed the microscopic structure of the liquid-like clusters constituting the domain based on the fluorescence lifetime measurement. We believe that our study will be an important milestone for the microscopic study of laser trapping-induced crystallization and large dense domain formation.
URI: http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT070352515
http://hdl.handle.net/11536/139304
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