JACS: far red light activated BODIPY for living cell super-resolution imaging

The appearance of super-resolution imaging promotes the development of fluorescent probes These fluorescent probes with excellent optical properties can be applied to the visualization of living cell super-resolution At present, most of the synthesized fluorescent dyes are cyanine dyes and oxaanthracene derivatives The chromophore of BODIPY is rarely used The imaging principle is based on the light-induced quenching agent disconnection However, the poor fluorescence contrast produced by this mechanism complicates the detection at the single molecule level Therefore, it has become a research hotspot to design a new photochemical mechanism of BODIPY dye for super-resolution visualization of living cells Recently, Professor Fran ç ISCO M raymo of the University of Miami and Professor Hao F Zhang of Northwestern University designed a new photochemical mechanism to make the wavelength of BODIPY chromophore red shift and allow the selective excitation of the product Based on the above principle, the author designed and synthesized a series of photo activated BODIPY derivative fluorophores, which are composed of BODIPY chromophore, photo conversion chromophore and styrene group covalently (Fig 1) On the basis of spectroscopic characterization, the fluorophore was further used in COS-7 cell imaging Relevant achievements were published on J am Chem SOC (DOI: 10.1021 / JACS 8b09099) under the title of "far red photoactivated bodies for the super resolution imaging of live cells" Fig 1 light induced conversion mechanism (1, 3, 5 converted to 2, 4, 6, respectively) (source: J am Chem SOC.) the author selected compound 1 as the study object, studied its absorption and emission spectra in tetrahydrofuran solution, and conducted time-dependent study under light irradiation (Fig 2) The results show that compound 1 is transformed into compound 2 after being irradiated by light When the excitation wavelengths of compound 1 and compound 2 are 565 nm and 620 nm respectively, the absorption and emission spectra have significant red shifts The change of absorption spectra with time under light irradiation is more intuitively described Fig 2 standard absorption and emission spectra of compound 1 (a, c); standard absorption and emission spectra of compound 2 (B, d) The change of absorption spectrum (E) of compound 1 under 350 nm light (source: J am Chem SOC.) subsequently, the author monitored the photoactivation of compound 1 on PMMA film by multi spectral photon location imaging (SPLM) and collected the spatial and spectral information of single molecule emission behavior It was found that compound 1 showed very weak fluorescence under 642 nm excitation light When compound 1 was activated under 405 nm excitation light and placed under 642 nm excitation light, it produced strong fluorescence signal PMMA film more intuitively shows the process from photoactivation to photobleaching of compound 2 (Fig 3b) The Gauss fitting curve and scatter diagram show that the environmental heterogeneity of the matrix in different regions is the cause of the spectral heterogeneity (Fig 3C and Fig 3D) Fig 3 emission spectrum (a) of compound 1, photoactivation process on PMMA membrane (b), Gauss fitting curve and scatter diagram (C, d) (source: J am Chem SOC.) further, the author selected COS-7 cell line to study the horizontal imaging and organelle localization ability of compound 5 cell The imaging results show that strong fluorescence signal can be observed under 561 nm excitation light, and the fluorescence signal of compound 5 overlaps with lysosomal fluorescence signal (Fig 4) Based on the above results, we conclude that compound 5 has good cell imaging ability and lysosomal localization ability Fig 4 laser confocal imaging of compound 5 co incubated with COS-7 cells (source: J am Chem SOC.) finally, the transformation and regeneration process and localization accuracy of compound 5 in a single lysosome were studied Confocal imaging showed that compound 5 could complete the photoactivation process in a single lysosome (Fig 5a-c) The scintillation pattern of compound 5 in a single lysosome showed that compound 5 showed a significant high-intensity peak over the whole collection time and lasted for a long time (Fig 5d) The single lysosome in Figure 5B and 5C was fitted by Gauss, and it was proved that the size of single lysosome was about 80 nm (Figure 5e) The author found that the positioning accuracy of compound 5 was 15 nm and the average photon number was about 2000 (Fig 5F and Fig 5g) The above results show that compound 5 can visualize a single lysosome for a long time and accurately Fig 5 compound 5 in a single lysosome imaging, scintillation track map, Gaussian fit curve and lysosome positioning accuracy map (source: j.am Chem SOC.) all in all, the author designed and synthesized a series of light activated BODIPY derivative fluorescence clusters, which can super-resolution, long-term positioning imaging of lysosomes, positioning accuracy of 15 nm This photoactivated fluorophore has potential applications in the field of single molecule tracking and localization microscopy of living cells.