Studies on synthesis and properties of novel fluorescent molecules and their applications to super-resolution bioimaging

Abstract

In recent years, super-resolution microscopy has become an indispensable method for detecting cellular metabolism and identifying biological signals concomitantly. However, the super-resolution technique induces rapid quenching on live-cell imaging by strong laser power or long exposure time. Therefore, the development of novel fluorophores for super-resolution microscopy is essential for advanced research since an appropriate probe determines the potential of the imaging. Specifically, photostability is a crucial parameter for fluorescent probes for super-resolution since it enables continuous imaging.

Herein, we report photostable NTD-based fluorescent probes that are applicable for cell organelle targeting such as mitochondria and lysosomes. In Chapter 2, novel fluorescent probes based on the NTD core were designed and synthesized for cell organelle targetable probes for super-resolution bioimaging. By extending the conjugation of the NTD core, we synthesized fluorophores based on NTD (NTDT, NTDPy, NTDT-DCV) having various color spectra such as blue, green, orange, and red. The photophysical properties of NTD-based fluorophores have been studied in detail and they showed high brightness and high absorption coefficient at the imaging wavelength. It means that they can minimize phototoxicity by decreasing excitation light intensity and exposure time while imaging.

In Chapter 3, we directly examine the cell-organelle targeting bioimaging of Confocal laser scanning microscopy (CLSM) and high-resolution methods. After the screening step of the compounds synthesized in Chapter 2, we selected two lysosome targeting probes, NTD-Mor, NTDT-Mor, and two mitochondria targeting probes, NTDT-TPP, and NTDT-DCV-TPP that show great possibility under the cell environment. These probes have selective affinity for recognizing with a specific cell organelle. As a result, NTD-based fluorophores have high photostability, selectivity, cell viability at confocal microscopy. We also report that the NTD-based fluorophores selectively binding to targeting mitochondria and lysosomes.

Moreover, we report super-resolution imaging by combining NTD-based fluorescent probes of various colors such as NTD (blue) and NTDT (green), and NTDT-DCV (red). Furthermore, the green fluorophore, NTDT-TPP, and NTDT-Mor exhibit outstanding resistance to photobleaching by depletion laser of STED (Stimulated Emission Depletion). Indeed, we succeeded in attaining super-resolution imaging at SIM (structured illumination microscopy) without quenching at NTD-based fluorescent dyes, meaning that all they could endure long exposure radiation time without rapid quenching. Therefore, compared to CLSM images, STED imaging and SIM imaging increased resolution. As a result, the NTD-based fluorophores demonstrated outstanding quality images of photostability, selectivity, and viability for super-resolution imaging.