Aberration-corrected cryogenic objective mirror with a 0.93 numerical aperture

Fujiwara, Masanori; Ishii, Takaki; Ishida, Kunio; Toratani, Y.; Furubayashi, Taku; Matsushita, Michio M.; Fujiyoshi, Satoru

doi:10.1063/1.5110546

Cited by 8 publications

(4 citation statements)

References 19 publications

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“…The dynamic properties of protein have been proposed to be significant for highly efficient photoelectric conversion in the RC. − Although we discussed here the energy transfer process in the hRC, the individual Chl a -A 0 is also available as a probe of the charge separation and electron transfer reaction occurring on each branch. ,, Additionally, the spectral peak of Chl a -A 0 is clearly separated from that of antenna pigment Bchl g s, permitting the spectral correlation analysis to reveal the relationship among protein dynamics around each chromophore . The recently developed objective lens with NA = 0.93 can facilitate the single-molecule spectroscopy of Chl a -A 0 and the spectral analysis. Therefore, the single-molecule approach will offer a chance to understand the molecular mechanism of how protein dynamics are controlled in the RC and consequently regulate the RC functions.…”

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confidence: 99%

“…20,43,44 Additionally, the spectral peak of Chl a-A 0 is clearly separated from that of antenna pigment Bchl gs, permitting the spectral correlation analysis to reveal the relationship among protein dynamics around each chromophore. 45 The recently developed objective lens with NA = 0.93 46 can facilitate the single-molecule spectroscopy of Chl a-A 0 and the spectral analysis. Therefore, the singlemolecule approach will offer a chance to understand the molecular mechanism of how protein dynamics are controlled in the RC and consequently regulate the RC functions.…”

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confidence: 99%

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

Kondô

Mutoh

Tabe

et al. 2020

J. Phys. Chem. Lett.

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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 as. The chlorophyll serves as the primary electron acceptor (Chl a-A 0 ) responsible for light harvesting and charge separation. The single-molecule spectroscopy of Chl a-A 0 can be used to investigate heterogeneities of the RC photochemical function, though the low fluorescence quantum yield (0.1%) makes it difficult. Here, we show the fluorescence excitation spectroscopy of individual Chl a-A 0 s in single hRCs at 6 K. The fluorescence quantum yield and absorption cross section of Chl a-A 0 increase 2-and 4-fold, respectively, compared to those at room temperature. The two Chl a-A 0 s in single hRCs are identified as two distinct peaks in the fluorescence excitation spectrum, exhibiting different excitation polarization dependences. The spectral changes caused by photobleaching indicate the energy transfer across subunits in the hRC.

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

Kondô

Mutoh

Tabe

et al. 2020

J. Phys. Chem. Lett.

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“…However, the major problem with the single-component configuration was the lack of a high-NA objective that works in superfluid helium. Since 2004, we have been developing nine reflecting objectives that work in superfluid helium (cryo-objective mirrors). − The cryo-objective mirrors consist of two mirrors. To maintain the optical alignment when cooling down to a few K, the objective mirrors were made of single-piece fused silica, and the two mirrors were produced by vacuum-deposition of aluminum onto the polished fused-silica surfaces.…”

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confidence: 99%

Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami

et al. 2020

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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. We performed the localization nanoscopy of these two fluorophores at 1.8 K with a far-field fluorescence microscope. Under the cryogenic conditions, the fluorophores were perfectly immobilized and their photobleaching was drastically suppressed; consequently, the lateral spatial precision (a measure of reproducibility) was increased to 1 nm. However, the lateral spatial accuracy (a measure of trueness) remained tens of nanometers. We observed that the fluorophore centroids were laterally shifted as a function of the axial position. Because the orientation of the transition dipole of the fluorophores was fixed under cryogenic conditions, the anisotropic emission from the single fixed dipole had led to the lateral shift. This systematic error due to the dipole-orientation effect could be corrected by the three-dimensional localization of the individual fluorophores with spatial precisions of (lateral) 1 nm and (axial) 17 nm. In addition, the xy-error arising from the three-dimensional (3D) orientation of the scaffold with the two fluorophores 11 nm apart was estimated to be 0.3 nm. As a result, the individual fluorophores on the DNA origami were localized at the designed position, and the lateral spatial accuracy was quantified to be 4 nm in the standard error.

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“…The key optical element is a cryoresistant objective mirror, which we called an INAGAWA mirror (Figures S1 and S2). We have been developing various cryo-objective mirrors − because most high-quality objective lenses do not function under cryogenic conditions. The INAGAWA mirror consists of spherical and aspherical mirrors, produced by the vacuum deposition of aluminum onto a single fused silica substrate.…”

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

Furubayashi

Ishida

Kashida

et al. 2019

J. Phys. Chem. Lett.

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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). Although each fluorophore was localized with a 1 nm lateral precision by acquiring an image at one axial position within the focal depth of ±0.7 μm, the distance between the two fluorophores on the lateral plane (D xy ) was distributed from 0 to 50 nm. This systematic error was mainly due to detecting with the large focal depth the dipole emission from orientationally fixed fluorophores. Each fluorophore was localized with precisions of ±1 nm (lateral) and simultaneously ±11 nm (axial) by acquiring images every 100 nm in the axial direction from −900 to 900 nm. By correcting the dipole orientation effects, the distribution of D xy was centered around the DNA length. The average and standard deviation of D xy were 10 and 5 nm.

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Aberration-corrected cryogenic objective mirror with a 0.93 numerical aperture

Cited by 8 publications

References 19 publications

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

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

Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami

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

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