Cryogenic Single-Molecule Fluorescence Detection of the Mid-Infrared Response of an Intrinsic Pigment in a Light-Harvesting Complex.

We observed the mid-infrared (MIR) response of a single pigment of bacteriochlorophyll at the B800 binding site of a light-harvesting 2 complex. At a temperature of 1.5 K, a single complex in a spatially isolated spot in a near-infrared (NIR) fluorescence image was selected and was simultaneously irradiated with MIR and NIR light.

Superfluid helium nanoscope insert with millimeter working range.

A superfluid helium insert was developed for cryogenic microscopy of millimeter-sized specimens. An optical-interferometric position sensor, cryogenic objective mirror, and piezo-driven cryogenic stage were fixed to an insert holder that was immersed in superfluid helium. The single-component design stabilized the three-dimensional position of the sample, with root-mean-square deviations of (x, lateral) 0.

Energy transfer fluctuation observed by single-molecule spectroscopy of red-shifted bacteriochlorophyll in the homodimeric photosynthetic reaction center.

The photosynthetic reaction center of heliobacteria (hRC) is a homodimeric chromoprotein responsible for light harvesting and photoelectric conversion. The fluorescence of the hRC is radiated from a bacteriochlorophyll (Bchl) g having the lowest energy level, called red-Bchl g. The homodimeric architecture of the hRC indicates that it includes two red-Bchls g arranged symmetrically in pairs.

Variable immersion microscopy with a high numerical aperture.

Three-dimensional (3D) optical microscopy with a high numerical aperture (NA) remains challenging for thick biological specimens owing to aberrations arising from interface refractions. We developed a variable immersion lens (VIL) to passively minimize these aberrations. A VIL is a high-NA concentric meniscus lens and was used in combination with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror).

Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami.

Far-field fluorescence localization nanoscopy of individual fluorophores at a temperature of 1.8 K was demonstrated using DNA origami as a one-nanometer-accurate scaffold. Red and near-infrared fluorophores were modified to the scaffold, and the fluorophores were 11 or 77 nm apart.

Cryogenic Single-Molecule Spectroscopy of the Primary Electron Acceptor in the Photosynthetic Reaction Center.

The photosynthetic reaction center (RC) converts light energy into electrochemical energy. The RC of heliobacteria (hRC) is a primitive homodimeric RC containing 58 bacteriochlorophylls and 2 chlorophyll s. The chlorophyll serves as the primary electron acceptor (Chl -A) responsible for light harvesting and charge separation.

Nanometer Accuracy in Cryogenic Far-Field Localization Microscopy of Individual Molecules.

We demonstrate the nanometer accuracy of far-field fluorescence localization microscopy at a temperature of 1.8 K using near-infrared and red fluorophores bonded to double-stranded DNA molecules (10.2 nm length).

Cryogenic Fluorescence Localization Microscopy of Spectrally Selected Individual FRET Pairs in a Water Matrix.

We prepared a pair with a visible-absorbing donor dye and a near-infrared fluorescing acceptor dye. The donor and the acceptor were covalently linked close enough for Förster resonance energy transfer to occur. Under cryogenic conditions at 1.

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.

Temperature-cycle microscopy reveals single-molecule conformational heterogeneity.

Our previous temperature-cycle study reported FRET transitions between different states on FRET-labeled polyprolines [Yuan et al., PCCP, 2011, 13, 1762]. The conformational origin of such transitions, however, was left open.

Reconstitution of bacterial photosynthetic unit in a lipid bilayer studied by single-molecule spectroscopy at 5 K.

As a model of photosynthetic unit (PSU), self-assembled aggregates of pigment-protein complexes from photosynthetic bacteria were prepared in a lipid bilayer by reconstitution of the light-harvesting 2 (LH2) complex and light-harvesting 1-reaction center (LH1-RC) complex through detergent removal of their micelles in the presence of lipids. By performing polarization-controlled fluorescence and fluorescence-excitation spectroscopy on single aggregates at a temperature of 5 K, the composition of individual aggregates was determined and excitation energy transfer (EET) between constituent complexes was observed. LH2 and LH1-RC from a bacterium, Rhodobacter (Rb.

Structural change of a cofactor binding site of flavoprotein detected by single-protein fluorescence spectroscopy at 1.5 k.

The visible fluorescence spectrum of single flavoprotein at a temperature of 1.5 K has been measured by one-photon excitation. The flavoprotein studied was a photoswitchable enzyme, photoactivated adenylyl cyclase.

Visible fluorescence spectroscopy of single proteins at liquid-helium temperature.

Fluorescence spectroscopy of single proteins at liquid-helium temperatures reveals a relation between structural dynamics and biological functions of the proteins. The technical difficulties in detecting visible fluorescence are chromatic aberration and optical background. They were overcome by a new optical design using reflective optics and employing two-photon excitation.

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.

Fourth-order Raman spectroscopy of wide-band gap materials.

Low-frequency surface vibrations were observed on a rutile TiO(2)(110) surface covered with trimethyl acetate (TMA) by using fourth-order Raman spectroscopy. The TMA-covered surface interfaced to air was irradiated with 18-fs light at a wavelength of 630 nm. A pump pulse excited vibrational coherence of Raman-active modes and a probe pulse interacts with the coherently excited surface to generate second harmonic light (315 nm), the intensity of which oscillated as a function of the pump-probe delay.

Molecular vibrations at a liquid-liquid interface observed by fourth-order Raman spectroscopy.

Interface-selective, Raman-based observation of molecular vibrations is demonstrated at a liquid-liquid interface. An aqueous solution of oxazine 170 dye interfaced with hexadecane is irradiated with pump and probe light pulses of 630-nm wavelengths in 17-fs width. The ultrashort pulses are broadened due to group velocity dispersion when traveling through the hexadecane layer.

Time-resolved infrared spectroscopy of K3Ta3B2O12 photocatalysts for water splitting.

Electrons photoexcited in K(3)Ta(3)B(2)O(12), an efficient photocatalyst for the water-splitting reaction driven by ultraviolet light, were observed using time-resolved IR absorption spectroscopy with microsecond resolution. When the catalyst was irradiated with 266 nm light pulses, a structureless absorption appeared at 3000-1500 cm(-1). The absorption was assigned to the optical transition of electrons that were band gap-excited and then trapped in mid-gap states.