Satoru Fujiyoshi

How Deep Is the Potential Well Confining a Protein in a Specific Conformation? A Single-Molecule Study on Temperature Dependence of Conformational Change between 5 and 18 K

The fluorescence excitation spectrum of a single chromophore molecule in a photosynthetic pigment-protein complex is known to change in time at liquid helium temperature. The spectral change reflects a conformational change of the protein to which the chromophore binds. This work follows the temporal behavior of the spectrum of a single chromophore in the temperature range between 5 adn 18 K. The temperature dependence reveals two types of conformational change of the protein, i.e., thermally activated motions over a potential barrier of ca. 0.1 kJ/mol and temperature-independent motions of tunneling of a proton.

Single-component reflecting objective for ultraviolet imaging and spectroscopy at cryogenic temperature

We have developed an objective for ultraviolet imaging and spectroscopy under cryogenic conditions. The objective was made of a single piece of silica with two spherical mirror surfaces. The pair of mirrors works as a reflecting objective for the optical rays that travel inside the silica. The extra refraction at liquid helium-silica interfaces was found to cause practically no chromatic aberration in the wavelength region from 360 to 980 nm. Using the objective with a focal length of 2 mm and a numerical aperture of 0.6, imaging an area of 130 μm×130 μm is possible with almost diffraction-limited quality.

Single-component reflecting objective for low-temperature spectroscopy in the entire visible region

A single-component reflecting objective was constructed for low-temperature spectroscopy with optimal imaging and transmission properties at all visible wavelengths. The performance of the objective immersed in superfluid helium at a temperature of 1.5K was tested by comparing dark-field images of uncolored polymer beads taken at wavelengths of 400 and 800nm. Under conditions optimized for imaging at both wavelengths, the size of the image is <1.3 times of the diffraction limit. The objective collects emission from a point source at focus with a solid angle of 0.32πsr.

Spectroscopy of single Pr3+ ion in LaF3 crystal at 1.5 K

Optical read-out and manipulation of the nuclear spin state of single rare-earth ions doped in a crystal enable the large-scale storage and the transport of quantum information. Here, we report the photo-luminescence excitation spectroscopy results of single Pr3+ ions in a bulk crystal of LaF3 at 1.5 K. In a bulk sample, the signal from a single ion at the focus is often hidden under the background signal originating from numerous out-of-focus ions in the entire sample. To combine with a homemade cryogenic confocal microscope, we developed a reflecting objective that works in superfluid helium with a numerical aperture of 0.99, which increases the signal by increasing the solid angle of collection to 1.16π and reduces the background by decreasing the focal volume. The photo-luminescence excitation spectrum of single Pr3+ was measured at a wing of the spectral line of the 3H4 → 3P0 transition at 627.33 THz (477.89 nm). The spectrum of individual Pr3+ ions appears on top of the background of 60 cps as isolated peaks with intensities of 20–30 cps and full-width at half-maximum widths of approximately 3 MHz at an excitation intensity of 80 W cm−2.

Reflecting microscope system with a 0.99 numerical aperture designed for three-dimensional fluorescence imaging of individual molecules at cryogenic temperatures

We have developed a cryogenic fluorescence microscope system, the core of which is a reflecting objective that consists of spherical and aspherical mirrors. The use of an aspherical mirror allows the reflecting objective to have a numerical aperture (NA) of up to 0.99, which is close to the maximum possible NA of 1.03 in superfluid helium. The performance of the system at a temperature of 1.7 K was tested by recording a three-dimensional fluorescence image of individual quantum dots using excitation wavelengths (λex) of 532 nm and 635 nm. At 1.7 K, the microscope worked with achromatic and nearly diffraction-limited performance. The 1/e2 radius (Γ) of the point spread function of the reflecting objective in the lateral (xy) direction was 0.212 ± 0.008 μm at λex = 532 nm and was less than 1.2 times the simulated value for a perfectly polished objective. The radius Γ in the axial (z) direction was 0.91 ± 0.04 μm at λex = 532 nm and was less than 1.4 times the simulated value of Γ. The chromatic aberrations between the two wavelengths were one order of magnitude smaller than Γ in each direction.

Three-Dimensional Localization of an Individual Fluorescent Molecule with Angstrom Precision

Among imaging techniques, fluorescence microscopy is a unique method to noninvasively image individual molecules in whole cells. If the three-dimensional spatial precision is improved to the angstrom level, various molecular arrangements in the cell can be visualized on an individual basis. We have developed a cryogenic reflecting microscope with a numerical aperture of 0.99 and an imaging stability of 0.05 nm in standard deviation at a temperature of 1.8 K. The key optics to realize the cryogenic performances is the reflecting objective developed by our laboratory. With this cryogenic microscope, an individual fluorescent molecule (ATTO647N) at 1.8 K was localized with standard errors of 0.53 nm (x), 0.31 nm (y), and 0.90 nm (z) when 106 fluorescence photons from the molecule were accumulated in 5 min.