Abstract:
Recently, the research group of Kai Li from the Department of Biomedical Engineering at the Southern University of Science and Technology, China collaborated with the research groups of Yiming Li and Xihan Chen to design and construct a type of spontaneous bioluminescent fluorescent dye for live cell labeling, used for single-molecule super-resolution imaging of cellular lipid droplets and lysosomes. This fluorescent dye exhibits a transformation between enol-keto isomers through spontaneous proton transfer in the ground state. Under 561 nm laser excitation, only a small amount of keto isomeric forms is excited to the bright state, while the enol isomeric forms remain in the dark state due to their inability to be excited, thus exhibiting spontaneous bioluminescence without the need for additional light activation conditions. This novel spontaneous bioluminescence mechanism, different from fluorescent dyes reported in the literature for single-molecule localization microscopy imaging, provides a new approach for developing fluorescent dyes with bioluminescent properties and offers a simple strategy for super-resolution imaging of subcellular structures in live cells.
Background:
Fluorescent dyes with fluorescence switch characteristics and organelle-targeting capabilities are indispensable for single-molecule localization microscopy techniques. However, effective design strategies are currently still limited by specific chemical frameworks, such as rhodamine and cyanine-derived structures. Furthermore, for precise localization, it is often necessary to rely on complex protein labeling tags, such as Halo-Tag and SNAP-Tag, to achieve specific labeling of fluorescent dyes for subcellular structures. Therefore, exploration into the development of novel fluorescent dyes with a new bioluminescence mechanism and direct targeting of organelles in live cell transmembrane staining appears particularly important.
Highlights:
(1) Enol-keto tautomeric fluorescent group and its bioluminescence mechanism
The authors synthesized two fluorescent groups, Lip-ML and Lip-PD, with proton transfer ability through aldehyde-amine condensation, as well as two non-proton transfer fluorescent groups, NH-ML and NH-PD (Figure 1a). In the UV absorption spectra, the absorption peaks of the enol state of Lip-ML and Lip-PD are located near 405 nm (Figure 1b). Fluorescence spectra show that the emission peaks of Lip-ML and Lip-PD are near 560 nm, while NH-ML and NH-PD exhibit almost no fluorescence emission, indicating the crucial role of proton transfer photochemical processes in radiation transitions (Figure 1c). Due to the enol-keto tautomerism, pure enol or pure keto isomeric forms cannot be obtained. To address this challenge, the authors first discovered the red-shifted absorption spectra of the keto isomeric form through Density Functional Theory (DFT) calculations, with absorption occurring at 561 nm, while the enol isomeric form shows almost no absorption (Figure 1d, 1g). The authors further used transient absorption spectra (TA) to detect the excited-state dynamics of Lip-ML and Lip-PD, confirming the dynamic conversion of enol-keto tautomerism in such molecules. It was found that under 400 nm laser excitation, two positive signals appear near 488 nm and 585 nm (<1 ps), and a negative signal appears near 412 nm. These two positive signals rapidly merge into one signal, while the negative signal remains unchanged (Figure 1e, 1h). At the same time, the authors observed that the dynamic time constant of the proton transfer process during the enol-keto tautomerism was 0.56 ps (Figure 1f, 1i).
(2) Single-molecule bioluminescence characteristics
Next, the authors characterized the single-molecule light-switching properties of the fluorescent dye. Using commercial dyes AF 647 and JF 549 as references, the authors extracted their single-molecule light-switching parameters, including photon count, switching cycle number, light-switching ratio (DC), and survival fraction (SF), demonstrating that the synthesized fluorescent dye possesses excellent reversible light-switching characteristics (Figure 2). This indicates its suitability for single-molecule localization microscopy imaging.
(3) Super-resolution imaging of live cell single-molecule localization
Finally, the authors performed super-resolution imaging of live cells. First, confocal microscopy was used for co-localization analysis of lipid droplets, with a Pearson correlation coefficient of 93.1%, indicating excellent targeting ability of the fluorescent dye to lipid droplets (Figure 3a). 3D single-molecule localization microscopy imaging showed the three-dimensional super-resolution structure of individual lipid droplets labeled with the fluorescent dye (Figure 3b, 3c). Single exponential fitting was also performed: the average photon count was 1974, localization precision was 10.1 nm, and FRC resolution was 36.4 nm (Figure 3d-f). Additionally, by changing the targeting groups of molecules, localization and super-resolution imaging of lysosomes in live cells could be achieved, demonstrating the potential of the spontaneous bioluminescence mechanism induced by enol-keto tautomerism in super-resolution imaging applications of various subcellular structures.
Conclusion and Outlook:
The authors report a novel class of easily synthesized fluorescent dyes with a new fluorescence switch mechanism for live cell transmembrane staining, utilized for single-molecule localization super-resolution microscopy imaging of subcellular structures. The authors innovatively validate that these molecules exhibit significant enol-keto tautomerism through spontaneous proton transfer in the ground state, ensuring bioluminescent performance by exciting a small amount of keto isomeric forms. The authors anticipate that this work will provide new insights for the biological applications of single-molecule localization super-resolution microscopy.
Article Details:
Facile Blinking Dyes with Spontaneous Enol-Keto Tautomerism for Super-Resolution Imaging of Subcellular Targets
Ji Gao†, Weijun Wu†, Lulu Zhou†, Shaokuan Gong, Chong Li, Xihan Chen*, Yiming Li*, and Kai Li*
DOI: 10.31635/ccschem.024.202303818
Article Link: https://doi.org/10.31635/ccschem.024.202303818