PASTEUR, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, Paris, France.
Sony Computer Science Laboratories, Paris, France.
Nat Commun. 2022 Mar 18;13(1):1482. doi: 10.1038/s41467-022-29172-0.
Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.
由于其灵敏度和多功能性，荧光广泛用于检测特定标记的生物分子。然而，荧光目前受到标签识别的限制，这是由于吸收/发射带的宽全宽和明亮荧光团的窄寿命分布。我们通过在不同光强下对可逆光开关荧光团（RSF）进行照明来引入额外的动力学维度来克服这一限制。在这个扩展的空间中，每个 RSF 的光化学反应动力学指纹具有无颜色失真的特点，仅用有限的硬件、出色的光子预算和最小的数据处理就可以恢复这个指纹。该指纹用于在不到 1 秒的时间内识别和区分 22 个光谱相似的可逆光开关荧光蛋白（RSFP）中的 20 个。该策略为扩展荧光成像的复用能力开辟了广阔的前景。
