Numerical Spherical Aberration Correction Method using Spatial Light Modulator under Deep-Part Fluorescence Observation
TL;DR: In this article, a liquid crystal on silicon spatial light modulator (LCOS-SLM) was used for correcting spherical aberration in a confocal fluorescence laser scanning microscopy (CFLSM) system.
Abstract: We have developed a confocal fluorescence laser scanning microscopy (CFLSM) incorporating a liquid crystal on silicon spatial light modulator (LCOS-SLM). To achieve high-resolution and high-contrast imaging for deeper part of the tissue with CFLSM, high numerical aperture objective lenses are required to tightly focus excitation light to meet Rayleigh limit(criterion) for the specimens. However, mismatch of refractive index at the boundary of interfacing materials, such as atmosphere, glass cover, and biological tissues, causes spherical aberration. Recently, we proposed a numerical method for correcting spherical aberration. In this method a pre-distorted wavefront pattern for aberration correction is calculated by ray tracing from a hypothetical focal point inside a specimen to the pupil plane. The resulting microscope can correct such spherical aberration. We observed 6.0μm fluorescent micro-beads dispersed three-dimensionally in agarose gel to confirm effectiveness of aberration correction. We reconstructed a three-dimensional image by taking 20 images by changing the depth with 1 μm interval and stacking them. It was apparent that the longitudinal/depth resolution was improved and that the intensity of fluorescence image was increased with aberration correction. While this method is applicable to other laser scanning microscopes, it has potential to enhance the signals for various super-resolution microscopic techniques, such as stimulated- emission-depletion (STED) fluorescence microscopy.
References
More filters
TL;DR: A new type of scanning fluorescence microscope capable of resolving 35 nm in the far field is proposed, overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function.
Abstract: We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.
5,110 citations
01 Jan 1990
TL;DR: Methods for Three-Dimensional Imaging and Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen.
Abstract: Foundations of Confocal Scanned Imaging in Light Microscopy -- Fundamental Limits in Confocal Microscopy -- Special Optical Elements -- Points, Pixels, and Gray Levels: Digitizing Image Data -- Laser Sources for Confocal Microscopy -- Non-Laser Light Sources for Three-Dimensional Microscopy -- Objective Lenses for Confocal Microscopy -- The Contrast Formation in Optical Microscopy -- The Intermediate Optical System of Laser-Scanning Confocal Microscopes -- Disk-Scanning Confocal Microscopy -- Measuring the Real Point Spread Function of High Numerical Aperture Microscope Objective Lenses -- Photon Detectors for Confocal Microscopy -- Structured Illumination Methods -- Visualization Systems for Multi-Dimensional Microscopy Images -- Automated Three-Dimensional Image Analysis Methods for Confocal Microscopy -- Fluorophores for Confocal Microscopy: Photophysics and Photochemistry -- Practical Considerations in the Selection and Application of Fluorescent Probes -- Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy -- Confocal Microscopy of Living Cells -- Aberrations in Confocal and Multi-Photon Fluorescence Microscopy Induced by Refractive Index Mismatch -- Interaction of Light with Botanical Specimens -- Signal-to-Noise Ratio in Confocal Microscopes -- Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging -- Blind Deconvolution -- Image Enhancement by Deconvolution -- Fiber-Optics in Scanning Optical Microscopy -- Fluorescence Lifetime Imaging in Scanning Microscopy -- Multi-Photon Molecular Excitation in Laser-Scanning Microscopy -- Multifocal Multi-Photon Microscopy -- 4Pi Microscopy -- Nanoscale Resolution with Focused Light: Stimulated Emission Depletion and Other Reversible Saturable Optical Fluorescence Transitions Microscopy Concepts -- Mass Storage, Display, and Hard Copy -- Coherent Anti-Stokes Raman Scattering Microscopy -- Related Methods for Three-Dimensional Imaging -- Tutorial on Practical Confocal Microscopy and Use of the Confocal Test Specimen -- Practical Confocal Microscopy -- Selective Plane Illumination Microscopy -- Cell Damage During Multi-Photon Microscopy -- Photobleaching -- Nonlinear (Harmonic Generation) Optical Microscopy -- Imaging Brain Slices -- Fluorescent Ion Measurement -- Confocal and Multi-Photon Imaging of Living Embryos -- Imaging Plant Cells -- Practical Fluorescence Resonance Energy Transfer or Molecular Nanobioscopy of Living Cells -- Automated Confocal Imaging and High-Content Screening for Cytomics -- Automated Interpretation of Subcellular Location Patterns from Three-Dimensional Confocal Microscopy -- Display and Presentation Software -- When Light Microscope Resolution Is Not Enough:Correlational Light Microscopy and Electron Microscopy -- Databases for Two- and Three-Dimensional Microscopical Images in Biology -- Confocal Microscopy of Biofilms — Spatiotemporal Approaches -- Bibliography of Confocal Microscopy.
4,121 citations
"Numerical Spherical Aberration Corr..." refers background in this paper
...Confocal fluorescence laser scanning microscopy (CFLSM) is widely known conventional optical imaging technique to increase optical resolution and contrast of fluorescent images by scanning excitation focal spot two dimensionally inside the specimens [1]....
[...]
Journal Article•
TL;DR: In this paper, the authors proposed a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point spread function.
Abstract: We propose a new type of scanning fluorescence microscope capable of resolving 35 nm in the far field. We overcome the diffraction resolution limit by employing stimulated emission to inhibit the fluorescence process in the outer regions of the excitation point-spread function. In contrast to near-field scanning optical microscopy, this method can produce three-dimensional images of translucent specimens.
3,987 citations
Book•
01 Jan 2006
TL;DR: The third edition of a classic text in biological microscopy includes detailed descriptions and in-depth comparisons of parts of the microscope itself, digital aspects of data acquisition and properties of fluorescent dyes, the techniques of 3D specimen preparation and the fundamental limitations, and practical complexities of quantitative confocal fluorescence imaging as mentioned in this paper.
Abstract: This third edition of a classic text in biological microscopy includes detailed descriptions and in-depth comparisons of parts of the microscope itself, digital aspects of data acquisition and properties of fluorescent dyes, the techniques of 3D specimen preparation and the fundamental limitations, and practical complexities of quantitative confocal fluorescence imaging.
894 citations
TL;DR: In this paper, the diffraction problem for a planar interface between two isotropic and homogeneous materials with this interface perpendicular to the optical axis is solved in a rigorous mathematical manner, and it satisfies the homogeneous wave equation.
Abstract: The diffraction of electromagnetic waves for light focused by a high numerical aperture lens from a first material into a second material is treated. The second material has a different refractive index from that of the first material and introduces spherical aberration. We solve the diffraction problem for the case of a planar interface between two isotropic and homogeneous materials with this interface perpendicular to the optical axis. The solution is obtained in a rigorous mathematical manner, and it satisfies the homogeneous wave equation. The electric and magnetic strength vectors are determined in the second material. The solution is in a simple form that can be readily used for numerical computation. A physical interpretation of the results is given, and the paraxial approximation of the solution is derived.
434 citations
"Numerical Spherical Aberration Corr..." refers background in this paper
...However, a dielectric interface between the principle plane of an objective and the focal point, such as of air, a glass slip, or biological tissues, causes spherical aberration due to the refractive index mismatch [2,3]....
[...]