Ultrafast photoconversion of the green fluorescent protein studied by accumulative femtosecond spectroscopy.
Ultrafast photoconversion of the green fluorescent protein studied by accumulative femtosecond spectroscopy.
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通过累积飞秒光谱研究绿色荧光蛋白的超快光转换。
DOI：
10.1016/j.bpj.2008.11.049
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发表时间：
2009
影响因子：
3.4
通讯作者：
T. Brixner
中科院分区：
文献类型：
--
作者：
F. Langhojer;F. Dimler;G. Jung;T. Brixner
利用累积产物检测方法研究了T203V绿色荧光蛋白（GFP）在飞秒激发下通过脱羧进行的不可逆光转化，该方法使我们能够测量每脉冲低至ΔOD = 10−7吸光度变化的小转换效率。800 nm和400 nm脉冲激发的功率研究表明，激发到GFP发色团中性形式的更高状态可以非常有效地诱导光转换。单激发中性发色团是两步激发过程的共振中间体，导致有效的光转换。在时间分辨双色实验中，我们通过将中性发色团的激发步骤从进一步的激发步骤分离到反应状态来确定这两步过程的动力学。动力学表明，只有初始激发的中性发色团才能进一步激发到反应性较高的激发态，而荧光中间态则不能。对于GFP在双光子荧光显微镜中的应用，发现的光化学行为意味着显微镜中使用的高强度条件可以容易地导致光转换，并且必须注意避免不必要的光转换。
The irreversible photoconversion of T203V green fluorescent protein (GFP) via decarboxylation is studied under femtosecond excitation using an accumulative product detection method that allows us to measure small conversion efficiencies of down to ΔOD = 10−7absorbance change per pulse. Power studies with 800- and 400-nm pulse excitation reveal that excitation to higher states of the neutral form of the GFP chromophore induces photoconversion very efficiently. The singly excited neutral chromophore is a resonant intermediate of the two-step excitation process that leads to efficient photoconversion. We determine the dynamics of this two-step process by separating the excitation step of the neutral chromophore from the further excitation step to the reactive state in a time-resolved two-color experiment. The dynamics show that a further excitation to the very reactive higher excited state is only possible from the initially excited neutral chromophore and not from the fluorescent intermediate state. For applications of GFP in two-photon fluorescence microscopy, the found photochemical behavior implies that the high intensity conditions used in microscopy can lead to photoconversion easily and care has to be taken to avoid unwanted photoconversion.
影响因子：
15
作者：
Bell, AF;Stoner-Ma, D;Tonge, PJ
通讯作者：
Tonge, PJ
DOI：
10.1073/pnas.93.16.8362
发表时间：
1996-08-06
影响因子：
11.1
作者：
Chattoraj, M;King, BA;Boxer, SG
通讯作者：
Boxer, SG