PASTEUR, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 24, rue Lhomond, 75005, Paris, France.
Sony Computer Science Laboratories, Paris, France.
Chemphyschem. 2022 Dec 5;23(23):e202200295. doi: 10.1002/cphc.202200295. Epub 2022 Sep 21.
We introduce HIGHLIGHT as a simple and general strategy to selectively image a reversibly photoactivatable fluorescent label associated with a given kinetics. The label is submitted to sine-wave illumination of large amplitude, which generates oscillations of its concentration and fluorescence at higher harmonic frequencies. For singularizing a label, HIGHLIGHT uses specific frequencies and mean light intensities associated with resonances of the amplitudes of concentration and fluorescence oscillations at harmonic frequencies. Several non-redundant resonant observables are simultaneously retrieved from a single experiment with phase-sensitive detection. HIGHLIGHT is used for selective imaging of four spectrally similar fluorescent proteins that had not been discriminated so far. Moreover, labels out of targeted locations can be discarded in an inhomogeneous spatial profile of illumination. HIGHLIGHT opens roads for simplified optical setups at reduced cost and easier maintenance.
我们提出 HIGHLIGHT 作为一种简单而通用的策略，用于选择性地对与特定动力学相关的可逆光激活荧光标记进行成像。该标记受到大振幅正弦波照明，从而在更高的谐波频率下产生其浓度和荧光的振荡。为了使标记具有特异性，HIGHLIGHT 使用与浓度和荧光振荡幅度谐波频率共振相关的特定频率和平均光强度。通过相敏检测，从单次实验中同时获得多个非冗余共振可观测值。HIGHLIGHT 用于选择性地对四种光谱相似的荧光蛋白进行成像，这些蛋白迄今为止一直无法区分。此外，可以在不均匀的空间照明分布中丢弃目标位置之外的标记。HIGHLIGHT 为简化光学设置铺平了道路，降低了成本，易于维护。
