High-resolution three-dimensional imaging of large specimens with light sheet–based microscopy

Verveer, Peter J.; Swoger, Jim; Pampaloni, Francesco; Greger, Klaus; Marcello, Marco; Stelzer, Ernst H. K.

doi:10.1038/nmeth1017

Cited by 313 publications

(223 citation statements)

References 12 publications

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“…It has been shown that performing multi-view reconstructions with SPIM data can yield improved imaging in many situations [21][22][23]. However, for the system in this study this technique is not useful because the organ of interest is a small bundle of cells in a concavity on a much larger organism, and the range of angles from which useful views can be obtained is quite limited.…”

Section: Sample Rotation Optimizes Imaging Accessmentioning

confidence: 99%

4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies

Swoger

Muzzopappa

López‐Schier

et al. 2010

Journal of Biophotonics

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A study demonstrating an imaging framework that permits the determination of cell lineages during organogenesis of the posterior lateral line in zebrafish is presented. The combination of Selective Plane Illumination Microscopy and specific fluorescent markers allows retrospective tracking of hair cell progenitors, and hence the derivation of their lineages within the primodium. It is shown that, because of its superior signal‐to‐noise ratio and lower photo‐damaged properties, SPIM can provide significantly higher‐quality images than Spinning Disk Confocal technology. This allows accurate 4D lineage tracing for the hair cells over tens of hours of primordium migration and neuromast development. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Section: Sample Rotation Optimizes Imaging Accessmentioning

confidence: 99%

4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies

Swoger

Muzzopappa

López‐Schier

et al. 2010

Journal of Biophotonics

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“…In contrast, csiLSFM applies an obvious but stable method to create the interference pattern reaching the finest limit. Additionally, csiLSFM is capable of rotating the specimen and implementing multiple-views imaging to improve the axial resolution (41,42). Lower wavelengths can be used that work well with smaller molecules.…”

Section: Discussionmentioning

confidence: 99%

csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm

Chang

Meza

Stelzer

2017

Proc. Natl. Acad. Sci. U.S.A.

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Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are demonstrated. Images of beads prove a near-isotropic lateral resolution of sub-100 nm. Images of yeast endoplasmic reticulum show that coherent structured illumination (csi) LSFM performs with physiologically relevant specimens.he prime reason to use fluorescence light microscopy is the observation of live, thick, multicellular, and 3D specimens under close to natural conditions. Thus, one has to work with specimens that are mounted in an aqueous medium and not attached to or established close to a coverslip. In addition, the main issues in fluorescence microscopy, fluorophore bleaching, endogenous organic molecule phototoxicity, and solar-level intensities have to be addressed (1).Light-sheet-based fluorescence microscopy (LSFM) has emerged as one of the most valuable novel tools in developmental biology (1-5), plant biology (6), and 3D cell biology (7). In contrast to an epifluorescence arrangement, LSFM uses at least two independently operated microscope objective lenses. The lenses used in the excitation of the fluorophores are arranged at an angle of 90°rela-tive to those used for the detection of the 3D fluorophore density distribution. In addition, special optical arrangements ensure that only a thin planar section centered on the focal planes of the detection lenses receives light. Hence, optical sectioning (8, 9), which is not available in conventional fluorescence microscopy, as well as no phototoxicity and no fluorophore bleaching outside a small volume close to the focal plane, are intrinsic properties of LSFM. The energy required to excite the fluorophores when recording a 3D stack of images can be reduced by two to four orders of magnitude relative to wide-field, confocal, and two-photon fluorescence microscopy (10, 11). Modern cameras are used to record millions of pixels in parallel, i.e., tens to hundreds of images with subcellular resolution within a few seconds.Because the lateral resolution of LSFM is similar to that of a conventional epifluorescence microscope, superresolution techniques have been combined with LSF...

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“…Dans une MFL, le système de détection consiste en un microscope à épifluorescence classique : un objectif, un fil- exemples [24,25]). De plus, l'excellent rapport signal/bruit de la MFL permet l'utilisation simultanée de techniques de traitement d'images, comme la déconvolution, et la fusion des différentes vues [26]. Ces techniques de reconstruction 3D ont été utilisées avec succès sur des embryons de medaka [20], des embryons de poisson zèbre ( Figure 4B), des follicules ( Figure 4A et Vidéo 1 1 ) et des embryons de drosophile [19,24,25], des drosophiles adultes ( Figure 4D et Vidéo 2 1 ) et de cellules épithéliales sous forme de kystes ou de sphéroïdes [27].…”

Section: Une Approche Différente : La Microscopie De Fluorescence à Funclassified

“…En raison du flux de photons atteignant le détecteur, la MFL permet une acquisition avec un très bon rapport signal/bruit (de l'ordre de 1 000:1 en mode rapide), bien plus élevé que pour les microscopes confocaux ou biphotoniques. Un des autres avantages de la MFL est l'imagerie à grande gamme dynamique qui permet d'effectuer de la déconvolution pour accroître la résolution spatiale [26] et de quantifier de manière très précise la distribution de la fluorescence d'une protéine marquée. Par exemple, dans les expériences sur les embryons de poisson zèbre, les variations de l'intensité des noyaux cellulaires, dont l'ADN était marqué, ont permis de déterminer le niveau de compaction de l'ADN sans aucune sonde fluorescente supplémentaire [18] (Figure 4C).…”

Section: Signal Et Acquisitionunclassified

Microscopie de fluorescence à feuille de lumière

Girard

Forget

2011

Med Sci (Paris)

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La microscopie confocale à balayage laser CLSM (confocal laser scanning microscopy) Elle utilise un diaphragme placé devant le photodétecteur qui permet de rejeter la lumière parasite émise par les fluorophores des plans situés hors du plan focal [4]. Le diamètre réglable de ce diaphragme conditionne en grande partie l'épaisseur de la coupe optique et donc la résolution axiale. Le CLSM est devenu la technique de référence pour l'imagerie 3D haute résolution de systèmes biologiques allant de la protéine aux tissus et a trouvé de nombreuses applications cliniques. Toutefois, malgré des progrès récents, la microscopie confocale connaît encore des limites majeures. En effet, le diaphragme étant de taille très inférieure au champ d'observation, il faut effectuer un balayage point par point du champ pour reconstruire une image de l'objet étudié. Cette détection sélective et séquentielle limite considérablement la résolution temporelle (≥ 1 μs/pixel) et induit une dégradation rapide de l'échantillon par photoblanchiment, pho-

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High-resolution three-dimensional imaging of large specimens with light sheet–based microscopy

Cited by 313 publications

References 12 publications

4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies

4D retrospective lineage tracing using SPIM for zebrafish organogenesis studies

csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm

Microscopie de fluorescence à feuille de lumière

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