PHOTON FLUORESCENCE SPECT & DYNAMICS OF ENHANCED GREEN FLUORESCENT PROTEIN

光子荧光光谱

• 批准号: 6349397
• 负责人: AHMED A HEIKAL
• 金额: $ 3.44万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349397
• 关键词: bioengineering /biomedical engineering; biological products; biomedical equipment development; biomedical resource; lasers; technology /technique

One-photon fluorescence correlation spectroscopy (FCS) has been conducted on a number of fluorescent dyes for photobleaching dynamics characterization has been studied experimentally. Photobleaching quantum yield (?b) of rhodamine green, cy2, and fluorescein, following one-photon excitation ( 100-fs pulses and CW), has been characterized. While rhodamine green did not bleach under pulsed excitation, fluorescein underwent photobleaching (?b=8.4x10-6 and 1.0x10-5) following the CW- and pulsed-excitation; respectively. Similar measurements has been carried out on cy3, allophycocyanin, B-phycoerythrin, R-phycoerythrin, R-phycocyanin and rose bengal using CW-one-photon FCS. Comparison was made between the measured ?b in this work and others whenever possible. For example, ?b~7.0x10-5 of R-phycoerythrin is in a good agreement with Whiet and Stryer (1998) using a different technique (1.1x10-5). While the bleaching of cy3 was independent of the singlet oxygen, no photobleaching was obser ved in de-oxygenated rose bengal suggesting the involvement of triplet state and singlet oxygen in photobleaching dynamics. Preliminary measurements on mutant green fluorescent protein (EGFP) as a function of excitation wavelength (405 and 488 nm) as well as pH-value (5.8 and 11.0) of the buffer were also carried out. The triplet state lifetime (?T) and the intersystem crossing rate (kISC) of those fluorophores were also analyzed using the intensity-dependent FCS results.

单光子荧光相关光谱(FCS)已经被 对几种荧光染料进行了光漂白动力学研究 对其表征进行了实验研究。光漂白 罗丹明绿、Cy2和荧光素的量子产率(？B)如下 研究了单光子激发(100-飞秒脉冲和连续波)的特性。 而罗丹明绿在脉冲激发下不会漂白， 荧光素经过光漂白(？b=8.4x10-6和1.0x10-5) 分别采用连续波激励和脉冲激励；类似 已对Cy3、别藻蓝蛋白、 B-藻红蛋白、R-藻红蛋白、R-藻蓝蛋白和孟加拉玫瑰的应用 CW-单光子FCS。并对测量的？B进行了比较。 只要有可能，这项工作和其他工作。例如，？b~7.0x10-5，共 R-藻红蛋白与Whiet和Stryer(1998)有很好的一致性 使用不同的技术(1.1x10-5)。而Cy3的漂白 与单线态氧无关，未观察到光漂白现象 在脱氧的孟加拉玫瑰中提示参与了三联体 光漂白动力学中的态氧和单态氧。初步 突变型绿色荧光蛋白(EGFP)的功能测定 激发波长(405和488 nm)以及pH值(5.8和 11.0)也进行了缓冲。三重态寿命 (？T)和这些荧光团的系间交叉率(KISC) 还使用强度依赖的FCS结果进行了分析。