To further understand biological systems, we use single molecule detection methods. By using single molecule detection methods, ensemble averaging can be avoided and, because of this, we can get accurate results of biological systems. Typically, the systems of interest are related to DNA, RNA and proteins in vitro. But we can observe these systems in vivo to understand the real-time dynamics of biological systems.
There are many useful methods to observe in ‘real’ systems. One of the methods used in our lab is ALEX-FRET.
As described in an upper side figure, we applied 3-color lasers to excite our samples. The three lasers are alternated which is used to our advantage for observing the systems of interest. With an alternating-laser system, fluorescence at each excitation time can be sorted to analyze the combination of fluorescence. By sorting the data from alternating-laser excitation experiments, we can define the stoichiometry values for each molecule. Because of the FRET phenomenon, we can measure not only the stoichiometry values but also information for the distance between two dyes. Moreover, we applied pico-molar(pM) concentrations of our sample to avoid ensemble averaging.
In brief, we are performing the experiments to investigate biological systems with ALEX-FRET at the single molecule level. Currently, we perform experiments for single nucleotide polymorphisms(SNPs) and enzymatic DNAs. Future plans include investigating biochemical systems with in vivo imaging techniques.
The benefit of single molecule measurement is observing the motion of “individual” molecules whereas bulk (ensemble) measurements only allow viewing of “average” properties. Through single molecule methods, it is possible to directly obtain information about heterogeneity of each molecule in ensemble, to track “true” time trajectories of single molecule, to detect multiple kinetic paths by transient intermediate states in reaction mechanisms and more. Among a variety of single molecule methods, TIRF (Total Internal Reflection Fluorescence) is a novel technique to study various dynamics of single molecules by observing the special thin part of cell-substrate region through emission of fluorophores attached on the surface. Since TIRF uses evanescent wave, which is formed when light is internally reflected off of an interface between specimen and glass(or qurtz) at an angle greater than the critical angle of total internal reflection, near the surface?within 100nm?it is possible to detect the events of thin cell surface through selective excitation of fluorophores in aqueous and cellular environments.
We study TIRF integrated with FRET (Fluorescence Resonance Energy Transfer) which is defined as a function of distance and energy transferred between a donor chromophore and an acceptor chromophore (in close proximity, typically <10nm) through nonradiative dipole-dipole coupling. In our experiments, we observe the real-time DNA replication by applying TIRF to a surface immobilized sample. DNA replication is the essential process of transferring genetic information of living organisms by copying DNA with the help of DNA polymerase. Among the factors relevant in this process, we can specifically investigate the dynamic motion of DNA polymerase. We demonstrate the effect of Mg2+ and dNTP on the replication rate through real-time monitoring of the DNA copying process which provides a glimpse of these complex dynamical motions involving DNA polymerase.
Microfluidics is the flow of fluids in a micro size volume. The system enables us to control the flow of fluids in both liquid and gas phases; which is called the laminar flow. The advantage of this small system is that once the channel is filled with the fluids, the channel is almost sealed and prevents a reaction with other factors such as oxygen or dust in the air.

The operation of microfluidics is dependent on a chip design. The channel structure is the major factor that determines the operation on the chip. It controls the flows of the chemicals on a single chip. This system can be operated under computer control, which reduces the time and requires less labor. Computer control also reduces the errors such as changing the reactant amount, splitting the chemicals, evaporation, or side reactions. This technique requires only a small amount of chemical materials, which can be a huge advantage when it is used for a single molecule technique. Accordingly, our team plans to combine the microfluidics technique with Total Internal Reflection Fluorescence (TIRF), which is one of the single molecule techniques.

In this group, chip material is PDMS and NOA. These are transparent in UV-visible wavelength light range; this property is powerful in combining microfluidics and fluorescence. Transparency is beneficial when using an optical tool such as fluorescence, which is commonly used in many chemical reactions. We can apply this system in Biology. Various cell environments can adapt the cell, and bring the responses of the cell in a short period of time in this system. We observe these responses of a cell, and obtain more information by using microfludics than other experiment. For example, we attach a cell on a micro-channel and insert the proteins or enzyme to flow. As we observe the flow, we get the information of a biological pathway in a cell. Our research is focused on DNA-RNA transcription and a membrane protein function. In vitro chemical control reaction experiment is advantageous to know the pathway factors. And this microfludics system will reduce the time and labor of observing the life pathways.

Figure A schematic microfluidics chip for small molecule detection in left and image of the real system for the operating droplet system in right side

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