Observing the structural dynamics of a single biomolecule is important in order to understand biological functions in vivo. However, the resolution of biological studies using standard far-field fluorescence microscopy, which is based on a lens and visible light, is limited by diffraction to ~λ/2NA (λ: wavelength of light, NA: numerical aperture of the objective lens). On the other hand, Stimulated Emission Depletion Nanoscopy (STED) breaks up the diffraction limit of conventional optical microscopy methods, and it is known as one of the innovative superresolution techniques in the life sciences. In other words, STED allows us to directly visualize the structural information of biological samples such as proteins and cells at the single molecule level. Fluorophores are excited from S1 to the higher excited state Sn by an excitation light, which resonates at the specific energy level of the fluorophores. Generally, fluorophores emit a red-shifted light spontaneously with the relaxation of the excited electrons to the ground state within several nanoseconds. If a STED beam is irradiated to the electrons in the excited state before the relaxation step, the electrons are stabilized by the stimulated emission and fluorescence is not observed, whereas fluorescence is observed when the STED beam is not used. Using this phenomenon, a circular excitation beam is coaligned with the STED beam to form a doughnut shape, and to produce a fluorescence focal spot that is smaller than a diffraction-limited spot. Therefore, the more the STED beam is enhanced, the smaller the induced fluorescence focal spot becomes. Moreover, STED nanoscopy can be used to monitor molecular dynamics.

Figure 1 formation of the superresolution of STED nanoscopy. (a) Mechanism of the formation of a focal spot by overlapping the excitation beam and the STED beam (b) The relationship between the shape and the power of STED beam

Currently, we are trying to establish a STED-based methodology to measure the length of DNA more accurately using HindⅢ-treated λDNA molecules as our model system. Furthermore, this technique will be applied to measure the distance between two defined sites within genomic DNA, specifically one that is hybridized with fluorescently labeled oligonucleotides. We are also making an effort to observe the interaction between biomolecules in cells, and to expand our research fields, which are relative to vital phenomena.

Figure 2 STED (left) and Confocal (right) image of 20 nm crimson beads

Sight plays a crucial role when recognizing the environment in nature. Therefore, collecting information that yields images of the targets is essential in understanding their own properties. Although many objectives can be observed without any special devices, some objectives with hundreds of micrometers or even smaller sizes cannot be observed by the naked eye. For this reason, people use special techniques called ‘microscopy’ to observe the small objectives and to obtain information on them.

There are three types of microscopy including electron microscopy (EM), scanning probe microscopy (SPM), and fluorescence microscopy. EM and SPM provide structural information on the targets in detail through high-resolution images (~10-10m). Despite the advantages of high-resolution images, they cannot be directly applied to biological research because EM uses an intense electron beam that can damage live cells, and SPM only provides information about the surface of the objective, but not on the interior of the objective. Fluorescence microscopy has relatively poor resolution (~10-7m) which is a result of the diffraction of light. However, it can bring out the image of the interior of living cells with molecular specificity. Because of these advantages, fluorescence microscopy is widely used to investigate biological problems.

The fluorescence microscopy system in our lab is based on confocal microscopy, which provides a diffraction limited resolution. The basic principle of fluorescence microscopy is observing labeled fluorophores in or on a sample through illumination. In confocal microscopy, a pinhole in front of the detector efficiently blocks the background signals that come from the non-focal planes (Figure 1-a). As a result, the signal-to-noise ratio is increased and the resolution of confocal microscopy is close to the diffraction limit (Figure 1-b).

Figure 1 (a) Schematic diagram of our confocal microscopy setup. (b) Confocal image of 50nm fluorescence nano diamond (FND). (c) Time-resolved fluorescence intensity measurement using acousto-optic tunable filter (AOTF). (d) Cross-correlation of fluorescence intensity fluctuation of Cy3 in water.

Our team built a setup for a fluorescent microscope. As a result, the accessibility of our setup is a significant advantage that can be applied to various kinds of research compared to a conventional microscope. Furthermore, we added some spectroscopy techniques on our microscopy setup. For example, we added an acousto-optic tunable filter (AOTF) to the pump-probe techniques, and a correlator to fluorescence correlation spectroscopy, respectively. Thus, we can obtain not only the image of a sample but also the photo-physical information (Figure 1-c, 1-d).

Our team investigated the properties of various fluorophores and developed a new method to break the diffraction limit of a far-field optical microscope. We are especially interested in the photo-switchable nature of carbocyanine dyes such as Cy5 and Alexa647, which are widely used to image biomolecules. We tried to overcome the disadvantages of some super-resolution microscopy techniques by using these photo-switchable characteristics of carbocyanine dyes.
We have been carrying out various experiments using JPK NanoWizard® Ⅰ BioAFM with BioCell™, ECCell™ and TAO™. Not only does AFM achieve molecular resolution but can operate in fluids permitting samples to be imaged in near-native conditions. Thus, there have been many studies of biological materials?such as nucleic acids and their complexes, membranes and living cells?using AFM in the few years since its inception.
The instrument is also capable of manipulating molecules and measuring the strength of molecular interactions with pico-newton sensitivity. With functionalized AFM tips, we can do single molecule force measurements. Moreover, it can be developed through combination with fluorescence microscopy, raman microscopy and other microscopy so that we can obtain a more detailed characterization--not only in the outfit images of molecules but in formation and decomposition of chemical bonding.
Our research interests are briefly categorized in two parts: “nano field” and “bio field”. In the nano field, the studies of electrochromic material like tungsten oxide and graphene are undertaken to investigate the mechanism of surface enhanced raman spectroscopy (SERS). In the bio field, we are currently working on observing the response differences to electric stimulation between normal cells and cancer cells. We hope that the information from these discrepancies will suggest a new approach to and direction for cancer therapy. In addition, we are doing further studies of histones and DNA-binding involved with DNA replication and DNA damage repair/recognition.

Single Molecule Spectroscopy
Super-resolution Optical & Nano-optical Nanoscopy
Real-time Femtosecond Dynamics