Development of Novel Super-resolution Nanoscopy and Spectroscopy Techniques by Using the Photophysical Characteristics of Fluorescent Dyes

Abstract

Fluorescence is a wonderful tool for investigation of the biomolecular structure and interactions as well as many of its non-biological applications, due to the noninvasive nature of light. Especially, the fluorescence microscope provides the ability to image the interior of living cells with molecular specificity. However, there exist a fatal disadvantage on the conventional fluorescence techniques called diffraction limit that restrict the spatial resolution of optical microscope to roughly half of wavelength (~250 nm for visible light), which is much larger than the size of biomolecules. Even in the diffusion-based single molecule spectroscopy, which can observe and analyze the individual molecules in ambient conditions to avoid the ensemble average, the diffraction limit harshly restricts the concentration of fluorescent molecules lower than 100 pM, where the biomolecular concentration in a living cell is usually much higher. In order to overcome the diffraction limit, super-resolution techniques have been developed in recent 20 years, mainly focused on the microscope. Although their specific strategies are slightly different from each other, the super-resolution techniques share an identical core mechanism, the switching between on and off states of target fluorophores. Nowadays, the super-resolution nanoscopy can image the three-dimensional structure of multiple components in living cells with < 20 nm spatial resolution, sometimes even with video-rate temporal resolution. However, the complex and expensive technical features hinder the common uses of super-resolution nanoscopy in wide range of applications. Hence we tried to improve and develop the super-resolution nanoscopy and spectroscopy techniques by utilizing the photophysics of fluorescent molecules. Reversible saturable optical fluorescence transition (RESOLFT) nanoscopy is a powerful method for super-resolution imaging of living cells with low light intensity. But the useful applications of RESOLFT nanoscopy is only performed with the fluorescent proteins, whose photobleaching resistance against the photoswitching cycles are much better than the organic fluorophores. We demonstrated for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores by precisely optimizing the imaging buffer and optical parameters to overcome the photobleaching problem. Using a covalently linked dye pair of Cy3 and Alexa647 labeled microtubules in a fixed HeLa cell, we achieved a spatial resolution of ~100 nm in the focal plane. This method provides a powerful alternative for biocompatible super-resolution imaging and also may open doors to optical writing and read-out with organic fluorophores. We also introduced a new, easily implementable sub-diffraction limit microscopy technique utilizing the optical AND-gate property of fluorescent nanodiamond (FND). We demonstrate that when FND is illuminated by two spatially-offset lights of different wavelengths, emission comes only from the region of their overlap, which is used to reduce the effective point spread function form ~300 nm to ~130 nm in lateral plane, well below the diffraction limit. With this technique, sub-diffraction limit microscopy can be implemented in a quick, easy, convenient, and inexpensive way with no technical complexities often encountered in other methods. Furthermore, since FND is an ideal fluorescent material with high photostability, this new method may find its use in dynamic imaging over a long duration of time. To overcome the concentration limit in diffusion-based single molecule techniques, we tried to combine the stimulated emission depletion (STED) nanoscopy with them, especially the alternating laser excitation (ALEX) spectroscopy utilizing the fluorescence resonance energy transfer (FRET). By spatially overlapping additional doughnut-shaped depletion beam with a focused light, we successfully reduced the effective observation volume in ALEX-FRET measurement, resulted to the observation of the individual molecules in higher concentration. We demonstrated the feasibility of this new technique, named ALFRED, by using a dual labeled single stranded DNA, and observed the single diffusion DNA molecules at up to 5 nM concentration, which is 100 times higher than that of typical single molecule measurement. Since a number of biomolecules have the dissociation constant in nanomolar concentration range, the ALFRED can offer the novel way for the single molecule spectroscopy in living cells.