We are interested in applying the experimental tools of physical chemistry to biological systems, trying to reveal the mechanisms underlying the dynamics therein. In particular, we employ a few types of fluorescence microscopy to study the dynamic aspects of live cells at the single-cell and single-molecule level. In doing so, we recently noted and demonstrated that certain novel nanoparticles with unique optical properties can be imaged in the cellular environment with huge advantages. As a result, we are witnessing such nanoparticles gradually replacing traditional fluorescent dye molecules and even quantum dots in the field of biological imaging. Given that the optical studies are pretty mature, the Holy Grails in this field will be conjugating biological molecules on the nanoparticle surface and specifically targeting cellular organelles. To this end, we are focusing our research efforts not only on the microscope development but also on the organic and inorganic wet chemistry. We also work on single-molecule spectroscopy of biologically important molecules such as DNA, RNA and proteins.
The nanoparticle system we chose in such endeavor is “upconverting nanoparticles” (UCNPs). UCNPs are nanoparticles where lanthanide ions (Yb3+ and Er3+) are doped in the host material such as NaYF4 nanocrystals (~30 nm in diameter).
They have unique optical properties, that is, they absorb near-IR (980 nm) photons and emit in the visible spectral range through a photophysical process called multi-photon “upconversion”. Because the efficiency of upconversion is very high, there is no need to use expensive femtosecond lasers providing strong laser pulses but a tiny and cheap CW diode lasers are good enough for the excitation light source. Obviously, the detection of visible emission from UCNPs does not require any “IR-grade” optics and cameras to be integrated in the equipment. Most importantly, even wide-field imaging without scanning of the laser or sample is feasible, so that “high quality”, “high-speed”, “real-time”, and “multi-photon” imaging can be achieved, which is unprecedented.
It is now very clear that the cytotoxicity of UCNPs is very low as confirmed by many research groups. Moreover, owing to the excitation in the near-IR range, the photo-damage of and autofluorescence from the biological samples are greatly suppressed. In short, UCNPs and near-IR excitation are combined to provide a perfect bio-friendly platform for live-cell imaging.
At the single-particle level, they turned out to be extremely photo-stable, namely, they exhibit neither photo-blinking nor photo-bleaching. Therefore, it is possible to image and track the nanoparticle for a long period of time without any interruption.
One of the greatest advantages of using UCNPs in biological imaging is that they don't require focused laser beams shining on them in order to be bright or luminescent. In other words, they can be imaged in the wide-field scheme. So rather than being scanned over the large filed of view, they are imaged by 2D projection of the laser beam. Therefore, the imaging speed can be as fast as video rate (~30 frames/sec), which is quite suitable for investigating fast biological dynamics. We have been establishing the UCNP-based wide-field multi-photon microscopy from 2008 and still in love with this remarkable photophysics, UPCONVERSION !!!!
Owing to the advatages of UCNPs for optical bio-imaging, one can also construct 3D images with high imaging rate. We are developing strategies to construct highly accurate 3D images of UCNPs in cells with finite thickness. The key to the "fast" and "background-free" 3D imaging is that (1) each section image is acquired by wide-field epifluorescence microscopy over the z-axis and (2) the autofluorescence from the cellular components is negligible. Only UCNPs satisfy these requirement perfectly among thousands of fluorophores developed so far. Recently, we performed the first 3D imaging of endocytosed UCNPs in living cells (HeLa), and even tracked the trajectories in real time.
As mentioned before, it is very clear that UCNPs do not bleach or blink upon a continuous excitation. Such photostability paradoxically poses a limit to their use as the probe for super-resolution imaging. The super-resolution imaging techniques such as STED (stimulated emission depletion), PALM (photo-activatd localization microscopy), and STORM (stochastic reconstruction microscopy) rely on photoblinking or photobleaching of the fluorophores to break the diffraction limit of optical microscopy. Thus, researchers are mostly looking for better ways to (1) decrease the laser power and/or (2) elongate the lifetime of the dyes, which have already been overcome by employing UCNP-based probes. All we have to do to achieve super-resolution with UCNPs are just designing a photo-switch to turn them on and off and making them stochastic in a contolled manner. To this end, we are focusing our efforts to testing various conditions for both the UCNP materials and laser systems.
In recent year, the research area of lighting emitting diode, biological imaging, temperature/pH-sensor requires novel luminescent materials which have high quantum yield. So, it is very important to synthesize luminescent materials and to find out unique photo-physical phenomena. We synthesize materials such as Oxide / Halide - Perovskite / Layered perovskite, Carbon quantum dots, and semi-conductor quantum dots using a synthesis method such as Hydrothermal synthesis, Solid state reaction and Hot injection synthesis. we are looking for novel luminescent matters with advanced luminescence properties.
From the “Puzzle of Rare-Earth Spectra”, Studies on the 4f-4f transitions of the lanthanide ion have continued. Upconversion materials are composed of “HOST CRYSTALS” with “LANTHANIDE IONS”. Because of Laporte forbidden properties of lanthanide ion electric dipole transitions, the effect of host crystal local symmetry around emitter ions on upconversion luminescence intensity (quantum yield) is very important. Crystal field induced Mixing of atomic configurations results in relaxation of parity forbidden selection rule of transitions. We are looking for hosts with various non-centrosymmetric sites and are studying the upconversion phenomenon through the process of doping lanthanides on such sites.