We applied three-dimensional super-resolution microscopy (3D-SRM) to fluorescence correlation spectroscopy (FCS). According to our predictions, this method can provide a spherical fluorescence spot area with atto-liter (10-18ℓ) volume, which is much smaller than that given by conventional FCS. Actually, the inflection point of the measured correlation function (CF) applied by 3D-SRM shifted to the shorter correlation time domain compared with that of conventional FCS. This result means that the effective fluorescence spot size becomes shrunken owing to 3D-SRM. This spot enables us to analyze diffusive motion of highly concentrated molecules in a small volume. It is expected that our proposed method can be applied to elucidating life phenomena as well as chemical dynamics.
In this study, a combined two-color phase plate (CTPP) was designed for super-resolution microscopy based on upconversion fluorescence depletion (FD), fabricated, and evaluated. It is composed of two types of phase plates, a spiral phase plate and an annular phase plate. A two-color phase plate modulates the phase of the erase beam while maintaining the phase of the pump beam. SRM performed using the proposed CTPP is expected to enable super resolution in both the focal plane and in the optical axis direction. Despite its complex structure, a highly accurate CTPP was obtained by using the exposure and etching processes used in semiconductor manufacturing.
Three-dimensional super-resolution microscopy based on fluorescence depletion (3D-SRM) was applied to the observation of immunostained microtubules having complicated structures stacking each other. Owing to a high depth resolution overcoming the diffraction limit, we can observe the tubulin fibers intertwined in a cell with improved image quality. Since the 3D-SRM system can be setup by adopting a simple two-color annular wave plate (TAWP) to a commercial laser scanning microscope (LSM), 3D-SRM is expected to be a powerful measurement method in life science.
We investigated lateral resolution in super-resolution microscopy based on fluorescence depletion, when an annular phase plate is applied to the erase beam. Applying a high NA the objective lens to microscopy, resolution becomes closer to that given by a spiral phase plate.
In super-resolution microscopy, we use fluorescence depletion, where an erase beam quenches a molecule in the S1 state generated by a pump beam, and then prevents fluorescence from the S1 state. When a tight doughnut shaped erase beam with is focused on the dyed sample together with a Gaussian pump beam, the remaining fluorescence spot in the focal plane becomes smaller than the diffraction-limited size. Applying destructive interference to the erase beam, erase beam has a minute three-dimensional dark spot surrounded by the light near the focal region. Since this spot introduces fluorescence depletion along the optical axis as in the focal plane, we can achieve three-dimensional super-resolution microscopy. However, to overcome the diffraction limit, an extremely precise optical alignment is required for projecting the focused pump beam into the dark spot of the erase beam. To resolve this technical issue, we fabricated a two-color annular hybrid wave plate (TAHWP) by combining two multi-order wave quartz plates. Although the pump and erase beams co-axially pass through the plate; the pump beam retains its original Gaussian shape, while the erase beam undergoes destructive interference. Inserting the TAHWP into a commercial scanning laser microscope, a three-dimensional spherical fluorescence spot with a volume of (~100 nm)3 can be created. Beside eliminating alignment problems and yielding a compact setup, the TAHWP makes our proposed method very suitable for commercial microscope systems. In this study, we report about detailed fabrication procedure and three-dimensional image properties given by the TAHWP.
KEYWORDS: Luminescence, Contrast transfer function, Super resolution microscopy, Super resolution, Point spread functions, Fluorescence spectroscopy, Spectroscopy, Diffraction, Spatial resolution, Rhodamine
We quantitatively investigated image properties in super-resolution microscopy using two-color fluorescence dip
spectroscopy. To evaluate the properties, the point spread function (PSF) and contrast transfer function (CTF) were
measured using a fluorescent scale together with a fluorescent bead. From the CTF, it has been found that visible light
can resolve a 100 nm line-and-space pattern by microcopy, and provide a contrast of 10%. The CTF corresponds to a
PSF with a FWHM of 130 nm. The value is two times finer than the diffraction limit size. An evaluation using a 100 nm
Φ fluorescent bead consistently supports the result given by the CTF for super-resolution microscopy. The measured
CTF shows that super-resolution microscopy can indeed improve the optical properties of fluorescent images and enable
us to observe a structure with a spatial resolution overcoming the diffraction limit.
The profile of the point spread function (PSF) in superresolution microscopy is studied theoretically. The fluorescence spot profile (i.e., the PSF) is determined by the focused beam patterns of the applied two-color lasers and the optical properties of the fluorescence-depletion process induced by the lasers (the pump and erase beams). In this study, the fluorescence-depletion process for the sample molecule is analyzed using a rate equation for a three-state model. Based on this result, we calculate the PSF for the case where the erase beam is modeled by a first-order Bessel function. In the case of an erase beam with a large photon flux, the obtained PSF has a Lorentzian-like shape, which seldom appears in traditional microscopy. In this work, we also investigated a possible relationship between the PSF and other parameters in the fluorescence-depletion process.
The formation of a doughnut-shaped laser beam is presented. To generate the beam, we use an optically addressed parallel-aligned nematic liquid-crystal phase spatial light modulator (PAL-SLM), and observe the shape of the focused beam. By using a compensating technique for wave aberration, the beam has a symmetric doughnut shape with a hole size of 1 µm on the focal plane. The experimental result shows that the generated beam can be expected to be applicable to super-resolving microscopy based on the fluorescence depletion process.
In order to investigate the technical feasibility of X-ray optical devises in the XANES energy region, we used typical carbon compounds with (pi) * molecular orbitals i.e. C60, C70, carbon nanotube and graphite. Their real and imaginary parts of the optical constants were studied. In this study, using XANES observation spectrum data and calculations by the Hartree-Slater method, the atomic-scattering factors of compounds near carbon K-edge region were obtained. It was found that outstanding anomalous dispersions are observed in the near carbon K-edge region; and it was also found that the compounds have peculiar and complicated optical properties due to the chemical-bond structures. Furthermore, based on the obtained results, we designed a metal/graphite normal-incident X-ray multilayer in the region and also considered its application to an X-ray microscopy.
We studied the UV and x-ray double-resonance absorption process in the case of 1 micrometers tryptohan. By adopting a three-state model for ultra-violet (UV) absorption we could calculate the excited molecular density. We also calculated the x-ray transmittance dependence on the UV-laser photon flux using x-ray absorption cross section obtained by a calculation based on the atomic Hartree-Slater method. We found that strong x-ray absorption occurs in the UV region with a photon flux of approximately 1025 photons/cm2/sec. Furthermore, we considered the feasibility of x-ray microscopy using double-resonance absorption. It is expected that only the images of selected molecules can be obtained with a good S/N.
A compact soft x-ray reflectometer usable in a small laboratory is developed for measurement of the soft x-ray reflectance of a multilayer mirror in a wide wavelength range, i.e. , including the water window (23 to 44Å). In this reflectometer, the reflectance can be measured as a function of wavelength or incidence angle. A laser-produced plasma soft x-ray source, which is small but has high brightness, makes it possible to construct the compact soft x-ray reflectometer. The soft x rays generated by the source are monochromatized with a grazing incidence reflection grating. Incidence angle dependency of the reflectance can be measured from 2 deg (almost normal incidence) to 85 deg. Reflectances of a Ni/Ti multilayer and a Mo/Si multilayer are measured, and 10.9% at a wavelength of 40.8Å and an angle of 75.1 deg and 38% at a wavelength of 135.5Å and an angle of 25.7 deg from normal incidence are obtained, respectively.
The design of the Schwarzschild objective considering an alignment error is discussed and its fabrication for a soft x-ray microscope is described. It is shown that performance degradation of a Schwarzschild objective by an alignment error can be reduced in a heterocentric system, in which the center of a curvature of a concave mirror is not coincident with that of a convex mirror. Objectives with a magnification of 100 and 224 have been fabricated. To complete an indispensable and difficult alignment within the objective itself, the two mirrors are mounted together in the same frame. The alignment errors of the mirrors are detected and their positions are corrected in a newly developed alignment system, which allows the alignment to achieve the tolerance of 0.3 micrometers . The fabrication of Mo/Si multilayers used for the mirrors are also described briefly. A reflectometer using a laser produced plasma source has been developed to measure reflectances of multilayers. The Mo/Si multilayer deposited by magnetron sputtering shows a reflectance over 40% at a wavelength of roughly 135 angstroms.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.