We introduce fluorescence-detected pump–probe microscopy by combining a wavelength-tunable ultrafast laser with a confocal scanning fluorescence microscope, enabling access to the femtosecond time scale on the micrometer spatial scale. In addition, we obtain spectral information from Fourier transformation over excitation pulse-pair time delays. We demonstrate this new approach on a model system of a terrylene bisimide (TBI) dye embedded in a PMMA matrix and acquire the linear excitation spectrum as well as time-dependent pump–probe spectra simultaneously. We then push the technique toward single TBI molecules and analyze the statistical distribution of their excitation spectra. Furthermore, we demonstrate the ultrafast transient evolution of several individual molecules, highlighting their different behavior in contrast to the ensemble due to their individual local environment. By correlating the linear and nonlinear spectra, we assess the effect of the molecular environment on the excited-state energy.

中文翻译：

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单分子超快荧光检测泵-探针显微镜

我们通过将波长可调的超快激光与共聚焦扫描荧光显微镜相结合，引入了荧光检测泵浦探针显微镜，从而能够在微米空间尺度上访问飞秒时间尺度。此外，我们从激发脉冲对时间延迟的傅立叶变换中获得光谱信息。我们在嵌入 PMMA 基质中的双酰亚胺 (TBI) 染料模型系统上展示了这种新方法，并同时获取线性激发光谱和时间相关的泵浦-探针光谱。然后我们将该技术推向单个 TBI 分子并分析其激发光谱的统计分布。此外，我们展示了几个单个分子的超快瞬态演化，由于各自的本地环境，突出了它们与整体的不同行为。通过关联线性和非线性光谱，我们评估了分子环境对激发态能量的影响。