Golmohamadi Farzin Ghane, Mehmood Amna, Phan Hoang Trong, Schmitt Franz-Josef, Laufer Jan
Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, von-Danckelmann-Platz 3, Halle (Saale) 06120, Germany.
Photoacoustics. 2025 Jun 6;44:100738. doi: 10.1016/j.pacs.2025.100738. eCollection 2025 Aug.
Pump-probe excitation of fluorophores has been shown to overcome the limitations of conventional multiwavelength imaging and linear unmixing approaches by providing fluorophore-specific contrast whilst eliminating the dominant background signal of endogenous chromophores. In this study, methods for generating pump-probe signals and images are investigated that rely on changing 1) the pump wavelength whilst keeping the probe wavelength fixed, 2) the probe wavelength whilst keeping the pump wavelength fixed, and 3) the time delay between the pump and probe pulse. Time-resolved PA signals were generated in purified solutions of genetically expressed red fluorescent proteins Katushka, mNeptune, and mCardinal in a cuvette. Spectra of the difference signal amplitude were found to correlate with the absorption and emission spectra. The difference signal plotted as a function of time delay also showed characteristic features for each protein. To demonstrate the capability of multiplexed imaging, the spatial distributions of Katushka and mNeptune were recovered from 2D difference images of a phantom. This study demonstrates that methods based on pump-probe excitation can be used to probe the photophysical properties of fluorophores. By detecting changes in these properties due to a stimulant, such as pH, the methods may find application in biosensing of the cellular microenvironment.
荧光团的泵浦 - 探测激发已被证明能够克服传统多波长成像和线性解混方法的局限性，它通过提供荧光团特异性对比度，同时消除内源性发色团的主要背景信号来实现这一点。在本研究中，我们研究了产生泵浦 - 探测信号和图像的方法，这些方法依赖于改变：1）在保持探测波长固定的同时改变泵浦波长；2）在保持泵浦波长固定的同时改变探测波长；3）泵浦脉冲和探测脉冲之间的时间延迟。在比色皿中，在基因表达的红色荧光蛋白Katushka、mNeptune和mCardinal的纯化溶液中产生了时间分辨光声信号。发现差分信号幅度的光谱与吸收光谱和发射光谱相关。绘制为时间延迟函数的差分信号也显示了每种蛋白质的特征。为了证明多重成像的能力，从体模的二维差分图像中恢复了Katushka和mNeptune的空间分布。本研究表明，基于泵浦 - 探测激发的方法可用于探测荧光团的光物理性质。通过检测由于刺激物（如pH值）导致的这些性质的变化，这些方法可能在细胞微环境的生物传感中找到应用。
