Experimental Biomolecular Physics, Dept. Applied Physics, Royal Institute of Technology (KTH), Albanova Univ Center, 106 91 Stockholm, Sweden.
Dept. Science and Technology, Linköping University, Campus Norrköping, 601 74 Norrköping, Sweden.
J Phys Chem B. 2023 Apr 13;127(14):3208-3222. doi: 10.1021/acs.jpcb.2c08016. Epub 2023 Apr 3.
Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.
近红外（NIR）荧光染料磺基花青 7（SCy7）的光致异构化动力学通过荧光相关光谱（FCS）和瞬态（TRAST）激发调制光谱的组合进行了研究。鉴定出发射峰红移的光致异构化态，动力学与三态光致异构化模型一致。将 TRAST 激发调制与光谱荧光法（光谱-TRAST）相结合，进一步证实了 SCy7 发射光谱中激发诱导的红移。我们展示了这种红光致异构化态如何影响 NIR 花青染料不同发射带中的闪烁动力学，以及它如何影响单分子、超分辨率以及Förster 共振能量转移（FRET）和多色读出。由于这种状态也可以在中等激发强度下存在，因此它也可以更广泛地影响荧光读出，也可以影响不依赖于高激发条件的读出。然而，在这项工作中鉴定和表征的这种额外的红光致异构化态及其光动力可以用作策略，将 NIR 花青染料的发射进一步推向 NIR 并增强具有吸收光谱的纳米粒子的光致敏化作用。最后，我们表明 SCy7 的光致异构化动力学及其红移光致异构体的形成强烈依赖于局部环境条件，如粘度、极性和空间位阻，这表明 SCy7 和其他 NIR 花青染料可用作环境传感器。这种环境信息可以在低自发荧光和散射条件下，通过 TRAST 在 NIR 中进行监测，并适用于广泛的样品和实验条件。
