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Journal ArticleDOI

Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination

TL;DR: Scanned Bessel beams are used in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets better suited to three-dimensional (3D) subcellular imaging.
Abstract: A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.
Citations
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Journal ArticleDOI
27 Aug 2015-Cell
TL;DR: It is proposed that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid- like compartments lie at the heart of ALS and, presumably, other age-related diseases.

1,988 citations


Cites methods from "Rapid three-dimensional isotropic i..."

  • ...The stress granules were imaged using a fast DSLM microscope with structured illumination (Planchon et al., 2011)....

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Journal ArticleDOI
24 Oct 2014-Science
TL;DR: A new microscope using ultrathin light sheets derived from two-dimensional optical lattices is developed, demonstrating the performance advantages of lattice light-sheet microscopy compared with previous techniques and highlighted phenomena that, when seen at increased spatiotemporal detail, may hint at previously unknown biological mechanisms.
Abstract: Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

1,585 citations


Cites background or methods from "Rapid three-dimensional isotropic i..."

  • ...S4) conceptually similar to ourearlierBesselmicroscope (7,8)....

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  • ...In response, we introduced (7) “nondiffracting”Bessel beams to light sheetmicroscopy (Fig....

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  • ...Specimens are cultured or mounted on 5-mmdiameter cover slips (Warner Instruments, 640700) cleaned before use according to our earlier protocol (7)....

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  • ...Certain aspects of the design are derived from our earlier Bessel beam work, so these earlier publications (7, 8, 57) remain valuable resources as well....

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  • ...This is about an order of magnitude more images per specimen than we achieved with linear Bessel beam plane illumination (7, 8) and nearly two orders of magnitude more than in typical confocal data sets of similar specimens at similar expression levels....

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Journal ArticleDOI
16 Jul 2015-Cell
TL;DR: The physical basis for light scatter in tissue is introduced, the mechanisms underlying various clearing techniques are described, and several of the major advances in light microscopy for imaging cleared tissue are discussed.

902 citations


Cites background from "Rapid three-dimensional isotropic i..."

  • ...…illumination microscopy (Holekamp et al., 2008), diSPIM (Wu et al., 2013), two-photon lightsheet (Planchon et al., 2011), Bessel beam lightsheet (Planchon et al., 2011), lattice lightsheet (Chen et al., 2014), and SCAPE (Bouchard et al., 2015), are limited to samples only a few hundreds…...

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  • ...…axial resolution, including objectivecoupled planar illumination microscopy (Holekamp et al., 2008), diSPIM (Wu et al., 2013), two-photon lightsheet (Planchon et al., 2011), Bessel beam lightsheet (Planchon et al., 2011), lattice lightsheet (Chen et al., 2014), and SCAPE (Bouchard et al., 2015),…...

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  • ..., 2011), Bessel beam lightsheet (Planchon et al., 2011), lattice lightsheet (Chen et al....

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  • ..., 2013), two-photon lightsheet (Planchon et al., 2011), Bessel beam lightsheet (Planchon et al....

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Journal ArticleDOI
TL;DR: Protocols spanning multiple dimensions of the CLARITY workflow are described, ranging from simple, reliable and efficient lipid removal without electrophoretic instrumentation to optimized objectives and integration with light-sheet optics (CLARITY-optimized light- sheet microscopy (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution.
Abstract: CLARITY enables the chemical transformation of intact biological tissues into a hydrogel–tissue hybrid. The hybrid samples can be interrogated using light and macromolecular labels, whilst retaining fine structure and native biological molecules.

776 citations

Journal ArticleDOI
TL;DR: Hydrogel-based structures can be built from within biological tissue to allow subsequent removal of lipids without mechanical disassembly of the tissue, creating a tissue-hydrogel hybrid that is physically stable, that preserves fine structure, proteins and nucleic acids, and that is permeable to both visible-spectrum photons and exogenous macromolecules.
Abstract: With potential relevance for brain-mapping work, hydrogel-based structures can now be built from within biological tissue to allow subsequent removal of lipids without mechanical disassembly of the tissue. This process creates a tissue-hydrogel hybrid that is physically stable, that preserves fine structure, proteins and nucleic acids, and that is permeable to both visible-spectrum photons and exogenous macromolecules. Here we highlight relevant challenges and opportunities of this approach, especially with regard to integration with complementary methodologies for brain-mapping studies.

676 citations

References
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BookDOI
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

Journal ArticleDOI
TL;DR: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide‐field fluorescence microscope with strikingly increased clarity compared to both conventional and confocal microscopes.
Abstract: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.

3,274 citations

Journal ArticleDOI
TL;DR: In this article, an investigation of the structure of the electromagnetic field near the focus of an aplanatic system which images a point source is made, and the results are illustrated by diagrams and in a tabulated form based on data obtained by extensive calculations on an electronic computor.
Abstract: An investigation is made of the structure of the electromagnetic field near the focus of an aplanatic system which images a point source. First the case of a linearly polarized incident field is examined and expressions are derived for the electric and magnetic vectors in the image space. Some general consequences of the formulae are then discussed. In particular the symmetry properties of the field with respect to the focal plane are noted and the state of polarization of the image region is investigated. The distribution of the time-averaged electric and magnetic energy densities and of the energy flow (Poynting vector) in the focal plane is studied in detail, and the results are illustrated by diagrams and in a tabulated form based on data obtained by extensive calculations on an electronic computor. The case of an unpolarized field is also investigated. The solution is riot restricted to systems of low aperture, and the computational results cover, in fact, selected values of the angular semi-aperture a on the image side, in the whole range 0 ≤ α ≤ 90°. The limiting case α → 0 is examined in detail and it is shown that the field is then completely characterized by a single, generally complex, scalar function, which turns out to be identical with that of the classical scalar theory of Airy, Lommel and Struve. The results have an immediate bearing on the resolving power of image forming systems; they also help our understanding of the significance of the scalar diffraction theory, which is customarily employed, without a proper justification, in the analysis of images in lowaperture systems.

2,636 citations

Journal ArticleDOI
13 Aug 2004-Science
TL;DR: In this article, a selective plane illumination microscopy (SPIM) was developed to generate multidimensional images of samples up to a few millimeters in size, which can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.
Abstract: Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.

2,152 citations

Journal ArticleDOI
TL;DR: Lifeact, a 17-amino-acid peptide, is described, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
Abstract: Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

2,036 citations