Single-molecule Fluorescence Techniques

For biological study, fluorescence microscopy is daily, but powerful tool due to its unique capability to visualize the specific component in the cell, tissue, or organism. In conventional fluorescence technique detects the fluorescence signal from the huge number of fluorescent molecule such as fluorescent protein or organic dye, but recent advance of related technologies makes the detection of fluorescence signal from single fluorescent molecule possible. The detection of single-molecule let us know unprecedent details about molecular dynamics and intermolecular or intramolecular interaction. In our lab, we has been tried to develop single-molecule fluorescence techniques, and studied biological problems using various single-molecule techniques

1. Single-molecule Multi-color FRET

In conventionalbiochemical studies, the results was obtained from huge number of nonsynchronized molecules. Therefore, detailed reaction step or molecular mechanism could not be understood. Single-molecule FRET provides us the unprecedent details about molecule interaction and dynamics, by reporting the distance change in nanometer scale with sub-nanometer spatial resolution and several millisecond temporal resolution.

However, the single-distance information obtained from conventional single-molecule FRET was not sufficient in many cases. To overcome this limitation we developed reliable single-molecule 3-color FRET by using proper fluorescent dye trio for the reliable detection of 3-color FRET. Although three distance information can be obtained due to the development of 3-color FRET, more information has been required for the study of complex biological system. So we developed more advanced, single-molecule 4-color FRET technique. And from that, we can obtain 6 distance information at once! In addition to that, we can know the correlation between two events separated more than conventional FRET range (> 10 nm) using dual FRET scheme, unique variation of 4-color FRET. We expect that many complex system can be understood by our multi-color FRET technique.

Figure 1 Single-molecule 4-color FRET result. 6 FRET efficiencies (right) of DNA Holliday junction (left) were determined from 4-color intensity time trace (middle).

2. Real-time confocal for single molecule studies in vivo

Line scanning confocal microscopy for single-moleculedetection in cell Huge background from other parts of cells makes the detection of single-molecule detection hard. Therefore, background reduction is one of most important thing for the detection of single-molecule in cell. Conventional wide-field imaging using epi-fluorescence microscopy was not successful for the detection of single-molecule due to its poor optical sectioning capability. TIRF (Total Internal Refection Fluorescence) microscopy can greatly reduce the background by limiting the excitation volume as near the surface (~ 100 nm from the surface) we can only observe the molecule at the surface. Confocal microscopy has good optical sectioning and can imaging at arbitrary depth, it takes too long time to obtain single image due to 2D scanning, and increasing of scanning speed caused the fluorescence saturation problem. To overcome this limitation of confocal microscopy, line scanning and spinning disk confocal microscopies were used, but commercially available such microscopies could not detect single-molecule signal.

We developed real-time confocal microscopy based on line scanning confocal microscopy. The use of line illumination instead of point enables the fast imaging at the video rate (~ 33 fps). For the detection of single-molecule, EMCCD (Electron Magnifying Charged Coupled Device) was used instead of previous line shape CCD. Optical sectioning capability of our setup was about 1.1 μm, showed best performance among the current wide-field single-molecule detection technique at arbitrary depth. This line scanning confocal microscopy can be used to any kind of study using single-molecule detection in cell with high background.

Figure 2 Schematic diagram of line scanning confocal microscopy

3. Fluorescence nanoscopy

Super-resolution imaging technique One of disadvantage of fluorescence technique is its resolution problem. Due to the diffraction limit of visible light, the resolution limit of fluorescence microscopy was about 200 nm, too large to distinguish the structure of small component of cell. Recently, super-resolution fluorescence imaging techniques which break the diffraction limit barrier was developed. STORM and PALM, these super-resolution imaging technique based on single-molecule localization and light-induced photo-activation. The resolution of these techniques was less than 50 nm, provided much more details by enhanced resolution of fluorescence image. In our lab, we aimed the development of these super-resolution techniques and tried to solve biological problem using this techniques.

Figure 3 Super resolution image result. Conventional fluorescence image (left) and STORM image (right) of COS-7 cell microtubule structure.

FRET combined with Mechanical manipulation

Not limited to observing motion of single-biomolecules, we can perturb molecular motion by using single-molecule manipulation tools: optical tweezers for stretching molecules, and magnetic tweezers for twisting them. Mechanical effect is emerging as an important factor in biology and lots of questions remain to be answered. We expect single-molecule FRET combined with optical and magnetic tweezers will be able to answer those questions.

4. optical trapping combined with multi-color FRET

Optical trapping provides well-controlled loads into biological molecules, allowing mechanical manipulation of individual molecules. These sole mechanical manipulation tools, however, do not provide an adequate spatial resolution for studying subtle conformational changes occurring at low force regime. We recently demonstrated that the problem can be solved by combining optical tweezers with single-molecule FRET. In this way, we could obtain the high spatial resolution of FRET while maintaining the accurate manipulationcapability of optical tweezers.

As biological systems become complex, however,the capability to monitor the correlated motion of more than three parts has been required. Previous attempts to combine with single-molecule FRET and optical tweezers could not monitor the correlated motion. To address the problem, we constructed a hybrid instrument combining single-molecule three-color FRET and optical tweezers. Using the technique, force-induced sequential unfolding of hairpin was investigated.

Figure . Schematics of Optical tweezers combined with multicolor FRET

5. Multiple Optical Traps combined with FRET

By splitting the laser beam spatially and temporally, we can make multiple optical traps with one laser source. First, we split the laser beams with polarization, and use one of them for a continuous and usual optical trap. The other polarized laser beam is separated spatially and temporally with AOD (Acousto-optic Deflector). We can move the continuous optical trap freely in 3 dimensional directions by moving lens on the computercontrolled translator. And, the time shared optical traps can be displaced on the focus plane by adjusting acoustic signal frequency to AOD.

With multiple optical traps, we can manipulate the DNA strands in a higher dimensional way. Our aim is to make the DNA knot and observe the DNA-binding enzyme behavior near the knot.

This MOT set-up is now combined with flow system and single-molecule fluorescence imaging set-up.

Four trapped beads (diameter: 1um, 3 time shared trap and 1 continuous trap)

6.  High-resolution Optical Trap combined with FRET

High Resolution Optical Tweezers

We combined FRET technique with high resolution optical tweezers. This technique will be one of the most promising single molecule techniques to investigate multi-dimensional dynamics of the complicated biomolecules.  Basically, combining optical tweezers with FRET technique can bare the whole picture of force spectroscopy correlated with internal conformational change of the biomolecules. Furthermore, this competitive ability can be highly enhanced by improving resolution of optical tweezers up to 3.4Å. This is achieved by time-shared dual trapping and differential detection techniques.

This technique will be applicable to study fine distinction of dynamics of enzyme substrate correlated with physical motion of the internal subunits as well as determination of multi-dimensional reaction landscape.

7.  Electro Magnetic Tweezer combined with FRET (including FRET + Magnetic Tweezer)

Convential mechanical magnetic tweezer (MT) allows the manupulation of bead along the direction of gravitational field only. To overcome this limitation, we are developing electromagntic tweezer. In electromagnetic tweezer, we can generate varioius shape of magnetic fields by controlling input currents. This allows the bead manupulation along the lethral direction.