Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution
13 Sep 2005-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 102, Iss: 37, pp 13081-13086
TL;DR: Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples, and a recently proposed method in which the nonlinearity arises from saturation of the excited state is experimentally demonstrated.
Abstract: Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arisesfromsaturationoftheexcitedstate.Thismethodcanbeused in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.
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TL;DR: This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.
Abstract: We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
7,924 citations
TL;DR: A high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores that can, in principle, reach molecular-scale resolution is developed.
Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.
7,213 citations
TL;DR: A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit, and suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.
3,437 citations
Cites background from "Nonlinear structured-illumination m..."
...Nonlinear structured illumination microscopy (10) has so far achieved resolution (full width at half-maximum (FWHM) of the point spread function (PSF)) of 50 nm and can in principle be improved further, but is limited by the photobleaching properties of the fluorophore, and uses excitation intensities near saturation, where photobleaching may be more pronounced than at low intensity (10)....
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...New imaging methods such as 4Pi microscopy (4–9), patterned illumination microscopy (10,11), stimulated emission depletion (STED) microscopy (12–14), and other types of reversible saturable optical fluorescence transition (RESOLFT) microscopies (15) can increase resolution by reducing the size of the observation volume or, correspondingly, by increasing the accessible Fourier space and therefore increasing the number of accessible spatial frequencies....
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...x 1⁄4 rmax rmin 1⁄4 k max A FA 1 k0 kBC k max x FB 1 kBC k0 ; (10)...
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TL;DR: Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
Abstract: In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.
2,730 citations
TL;DR: The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy.
Abstract: Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.
2,632 citations
References
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TL;DR: The second edition of this respected text considerably expands the original and reflects the tremendous advances made in the discipline since 1968 as discussed by the authors, with a special emphasis on applications to diffraction, imaging, optical data processing, and holography.
Abstract: The second edition of this respected text considerably expands the original and reflects the tremendous advances made in the discipline since 1968. All material has been thoroughly updated and several new sections explore recent progress in important areas, such as wavelength modulation, analog information processing, and holography. Fourier analysis is a ubiquitous tool with applications in diverse areas of physics and engineering. This book explores these applications in the field of optics with a special emphasis on applications to diffraction, imaging, optical data processing, and holography. This book can be used as a textbook to satisfy the needs of several different types of courses, and it is directed toward both engineers ad physicists. By varying the emphasis on different topics and specific applications, the book can be used successfully in a wide range of basic Fourier Optics or Optical Signal Processing courses.
12,159 citations
Book•
01 Jan 1968
TL;DR: The second edition of this respected text considerably expands the original and reflects the tremendous advances made in the discipline since 1968 as discussed by the authors, with a special emphasis on applications to diffraction, imaging, optical data processing, and holography.
Abstract: The second edition of this respected text considerably expands the original and reflects the tremendous advances made in the discipline since 1968. All material has been thoroughly updated and several new sections explore recent progress in important areas, such as wavelength modulation, analog information processing, and holography. Fourier analysis is a ubiquitous tool with applications in diverse areas of physics and engineering. This book explores these applications in the field of optics with a special emphasis on applications to diffraction, imaging, optical data processing, and holography. This book can be used as a textbook to satisfy the needs of several different types of courses, and it is directed toward both engineers ad physicists. By varying the emphasis on different topics and specific applications, the book can be used successfully in a wide range of basic Fourier Optics or Optical Signal Processing courses.
9,800 citations
TL;DR: The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation.
Abstract: Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
8,905 citations
TL;DR: Semiconductor nanocrystals prepared for use as fluorescent probes in biological staining and diagnostics have a narrow, tunable, symmetric emission spectrum and are photochemically stable.
Abstract: Semiconductor nanocrystals were prepared for use as fluorescent probes in biological staining and diagnostics. Compared with conventional fluorophores, the nanocrystals have a narrow, tunable, symmetric emission spectrum and are photochemically stable. The advantages of the broad, continuous excitation spectrum were demonstrated in a dual-emission, single-excitation labeling experiment on mouse fibroblasts. These nanocrystal probes are thus complementary and in some cases may be superior to existing fluorophores.
8,542 citations
Book•
01 May 1984
TL;DR: In this article, the authors present a general description of wave propagation in nonlinear media, including high-resolution nonlinear optical spectroscopy, and four-wave mixing and mixing.
Abstract: Introduction. Nonlinear Optical Susceptibilities. General Description of Wave Propagation in Nonlinear Media. Electrooptical and Magnetooptical Effects. Optical Rectification and Optical Field-Induced Magnetization. Sum-Frequency Generation. Harmonic Generation. Difference Frequency Generation. Parametric Amplification and Oscillation. Stimulated Raman Scattering. Stimulated Light Scattering. Two-Photon Absorption. High-Resolution Nonlinear Optical Spectroscopy. Four-Wave Mixing. Four-Wave Mixing Spectroscopy. Optical-Field-Induced Birefringence. Self-Focusing. Multiphoton Spectroscopy. Detection of Rare Atoms and Molecules. Laser Manipulation of Particles. Transient Coherent Optical Effects. Strong Interaction of Light with Atoms. Infrared Multiphoton Excitation and Dissociation of Molecules. Laser Isotope Separation. Surface Nonlinear Optics. Nonlinear Optics in Optical Waveguides. Optical Breakdown. Nonlinear Optical Effects in Plasmas. Index.
5,311 citations