雷射捕陷誘發溶菌酶叢集高濃度領域的螢光顯微分光研究

Abstract

We demonstrated a fluorescence microscopic study on formation of a large highly concentrated domain of hen egg white lysozyme (HEWL) liquid-like clusters by laser trapping. We prepared a HEWL buffer solution with a small amount of fluorescent dye-labeled HEWL molecules. A continuous wave near-infrared laser beam was used as a trapping light source and focused at a position 3 μm above a glass surface of the HEWL buffer solution for 30 min. When temporal change in fluorescence intensity in an area of 110 × 110 μm2 around the laser focus was monitored in real time under various experimental conditions, the intensity monotonously increased with the laser irradiation not only at the laser focus but also in the whole observation area. This result clearly means that a highly concentrated domain of the clusters is formed around the focus and the size is considerably larger than the focal size of about 1 μm. The further investigation of temporal change of the fluorescence intensity at a position 10 μm away from the focus showed us that laser trapping of 30 min laser irradiation produced two types of the cluster domains with different fluorescence intensity. We define these cluster domains with lower and higher fluorescence intensity as “Domain 1 and Domain 2”, respectively. In these domains, HEWL liquid-like clusters are connected with each other through hydrogen bonding interactions and form ordering and consequent rigid structure. The hydrogen bonding structure extends very rapidly up to a long distance. We systematically investigated laser polarization dependence of temporal change in the fluorescence intensity of these two domains. Interestingly, the depolarized laser irradiation produced the cluster domain with the highest fluorescence intensity. In order to gain an insight into ordering and rigidity of the clusters in two domains, we also investigated temporal change in the fluorescence intensity after stopping the laser trapping. The fluorescence intensity of Domain 1 after stopping laser trapping quickly decreased to almost an initial value for 20 min, while that of Domain 2 decreased much slowly and seemed to saturate at a certain high value for 2 hs. Surprisingly, compared to polarized laser irradiation, the fluorescence intensity in Domain 1 upon depolarized laser irradiation decreased considerably faster, in spite of the higher fluorescence intensity at 30 min of the irradiation. These results suggest that depolarized laser irradiation produces a more highly concentrated domain of the clusters with less ordering and rigidity. We also investigated growth behavior of a target HEWL crystal located in the domains with and after stopping laser trapping, and discuss dynamics and mechanism of formation and dissolution of the large highly concentrated domains of the clusters by comparing temporal change of fluorescence intensity and the crystal growth behavior.

We demonstrated a fluorescence microscopic study on formation of a large highly concentrated domain of hen egg white lysozyme (HEWL) liquid-like clusters by laser trapping. We prepared a HEWL buffer solution with a small amount of fluorescent dye-labeled HEWL molecules. A continuous wave near-infrared laser beam was used as a trapping light source and focused at a position 3 μm above a glass surface of the HEWL buffer solution for 30 min. When temporal change in fluorescence intensity in an area of 110 × 110 μm2 around the laser focus was monitored in real time under various experimental conditions, the intensity monotonously increased with the laser irradiation not only at the laser focus but also in the whole observation area. This result clearly means that a highly concentrated domain of the clusters is formed around the focus and the size is considerably larger than the focal size of about 1 μm. The further investigation of temporal change of the fluorescence intensity at a position 10 μm away from the focus showed us that laser trapping of 30 min laser irradiation produced two types of the cluster domains with different fluorescence intensity. We define these cluster domains with lower and higher fluorescence intensity as “Domain 1 and Domain 2”, respectively. In these domains, HEWL liquid-like clusters are connected with each other through hydrogen bonding interactions and form ordering and consequent rigid structure. The hydrogen bonding structure extends very rapidly up to a long distance. We systematically investigated laser polarization dependence of temporal change in the fluorescence intensity of these two domains. Interestingly, the depolarized laser irradiation produced the cluster domain with the highest fluorescence intensity. In order to gain an insight into ordering and rigidity of the clusters in two domains, we also investigated temporal change in the fluorescence intensity after stopping the laser trapping. The fluorescence intensity of Domain 1 after stopping laser trapping quickly decreased to almost an initial value for 20 min, while that of Domain 2 decreased much slowly and seemed to saturate at a certain high value for 2 hs. Surprisingly, compared to polarized laser irradiation, the fluorescence intensity in Domain 1 upon depolarized laser irradiation decreased considerably faster, in spite of the higher fluorescence intensity at 30 min of the irradiation. These results suggest that depolarized laser irradiation produces a more highly concentrated domain of the clusters with less ordering and rigidity. We also investigated growth behavior of a target HEWL crystal located in the domains with and after stopping laser trapping, and discuss dynamics and mechanism of formation and dissolution of the large highly concentrated domains of the clusters by comparing temporal change of fluorescence intensity and the crystal growth behavior.