High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics

AbstractLight-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 μm) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

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Dual-view light-sheet imaging through tilted glass interface using a deformable mirror

10.1101/2020.10.20.345306 ◽

2020 ◽

Author(s):

N Vladimirov ◽

F Preusser ◽

J Wisniewski ◽

Z Yaniv ◽

RA Desai ◽

...

Keyword(s):

Adaptive Optics ◽

High Speed ◽

Microfluidic Channel ◽

Microfluidic Devices ◽

Light Sheet ◽

Stochastic Gradient Descent ◽

Deformable Mirror ◽

Optical Aberrations ◽

Light Sheet Microscopy ◽

Gradient Descent Algorithm

AbstractLight-sheet microscopy has become one of the primary tools for imaging live developing organisms because of its high speed, low phototoxicity, and optical sectioning capabilities. Detection from multiple sides (multi-view imaging) additionally allows nearly isotropic resolution via computational merging of the views. However, conventional light-sheet microscopes require that the sample is suspended in a gel to allow optical access from two or more sides. At the same time, the use of microfluidic devices is highly desirable for many experiments, but geometric constrains and strong optical aberrations caused by the coverslip titled relative to objectives make the use of multi-view lightsheet challenging for microfluidics.In this paper we describe the use of adaptive optics (AO) to enable multi-view light-sheet microscopy in such microfluidic setup by correcting optical aberrations introduced by the tilted coverslip. The optimal shape of deformable mirror is computed by an iterative stochastic gradient-descent algorithm that optimizes PSF in two orthogonal planes simultaneously. Simultaneous AO correction in two optical arms is achieved via a knife-edge mirror that splits excitation path and combines the detection path.We characterize the performance of this novel microscope setup and, by dual-view light-sheet imaging of C.elegans inside a microfluidic channel, demonstrate a drastic improvement of image quality due to AO and dual-view reconstruction. Our microscope design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experimental conditions and high-content screening.

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Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

10.1101/243352 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tsung-Li Liu ◽

Srigokul Upadhyayula ◽

Daniel E. Milkie ◽

Ved Singh ◽

Kai Wang ◽

...

Keyword(s):

Adaptive Optics ◽

High Speed ◽

Developmental Stages ◽

Phenotypic Diversity ◽

Metastatic Cancer ◽

Light Sheet ◽

Intrinsic Variability ◽

Spatiotemporal Resolution ◽

Light Sheet Microscopy

AbstractTrue physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.One Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

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Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos

eLife ◽

10.7554/elife.40497 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 41

Author(s):

Mustafa Mir ◽

Michael R Stadler ◽

Stephan A Ortiz ◽

Colleen E Hannon ◽

Melissa M Harrison ◽

...

Keyword(s):

Transcription Factors ◽

Single Molecule ◽

High Speed ◽

Light Sheet ◽

Editorial Note ◽

Regulation Of Transcription ◽

Light Sheet Microscopy ◽

4D Imaging ◽

Active Transcription ◽

Drosophila Embryos

The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. Here, we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on our observations, we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

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Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination

Nature Communications ◽

10.1038/s41467-021-23249-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sota Takanezawa ◽

Takashi Saitou ◽

Takeshi Imamura

Keyword(s):

High Speed ◽

Bessel Beam ◽

Light Sheet ◽

Time Lapse ◽

Whole Body ◽

Wide Field ◽

Two Photon ◽

Light Sheet Microscopy ◽

Photon Excitation ◽

Two Photon Excitation

AbstractTwo-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600–1000 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2–3-μm axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with ~3 mm3 volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

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Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution

10.1101/240770 ◽

2017 ◽

Author(s):

Hao Wang ◽

Qingyuan Zhu ◽

Lufeng Ding ◽

Yan Shen ◽

Chao-Yu Yang ◽

...

Keyword(s):

Brain Mapping ◽

High Speed ◽

Large Scale ◽

Light Sheet ◽

Volumetric Imaging ◽

Tissue Sections ◽

Light Sheet Microscopy ◽

Cell Type Specific ◽

Microscopy Method ◽

Immunofluorescence Labeling

We describe a new light-sheet microscopy method for fast, large-scale volumetric imaging. Combining synchronized scanning illumination and oblique imaging over cleared, thick tissue sections in smooth motion, our approach achieves high-speed 3D image acquisition of an entire mouse brain within 2 hours, at a resolution capable of resolving synaptic spines. It is compatible with immunofluorescence labeling, enabling flexible cell-type specific brain mapping, and is readily scalable for large biological samples such as primate brain.

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