Langhojer Florian, Dimler Frank, Jung Gregor, Brixner Tobias
Institut für Physikalische Chemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.
Biophys J. 2009 Apr 8;96(7):2763-70. doi: 10.1016/j.bpj.2008.11.049.
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 DeltaOD = 10(-7) absorbance 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.
利用累积产物检测方法，在飞秒激发条件下研究了T203V绿色荧光蛋白（GFP）通过脱羧进行的不可逆光转换，该方法使我们能够测量低至每脉冲ΔOD = 10^(-7)吸光度变化的小转换效率。用800纳米和400纳米脉冲激发进行的功率研究表明，激发GFP发色团中性形式的更高能级会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，该过程会导致高效光转换。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与进一步激发到反应态的步骤分开，来确定这一两步过程的动力学。动力学表明，只有从最初激发的中性发色团而不是从荧光中间态才能进一步激发到非常活泼的更高激发态。对于GFP在双光子荧光显微镜中的应用，所发现的光化学行为意味着显微镜中使用的高强度条件可能容易导致光转换，必须注意避免不必要的光转换。
