scholarly journals Fast, multicolor 3-D imaging of brain organoids with a new single-objective two-photon virtual light-sheet microscope

2018 ◽  
Author(s):  
Irina Rakotoson ◽  
Brigitte Delhomme ◽  
Philippe Djian ◽  
Andreas Deeg ◽  
Maia Brunstein ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Three Dimensional ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Confocal Microscope ◽  
Large Field ◽  
Excitation Light ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Functional Features

ABSTRACTHuman inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies enlarge the scope of investigations to multi-cellular phenomena. A the highest level of complexity, brain organoids that – in many aspects – recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that belongs to the class of two-photon (2P) light-sheet microsocpes. Its particularity is that – unlike existing two- or three-lens designs – it is using a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petran spinning-disk scheme for achieving wide-field excitation. However, unlike the common Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This design, advantageous for 2P excitation circumvents problems arising with the tandem disk from the large wavelength-difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited in by the light sheet is collected by the same objective and imaged onto a fast sCMOS camera. We demonstrate three-dimensional imaging of TO-PRO3-stained embryonic bodies and of brain organoids, under control conditions and after rapid (partial) transparisation with triethanolamine and /ormamide (RTF) and compare the performance of our instrument to that of a confocal microscope having a similar numerical aperture. 2P-virtual light-sheet microscopy permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

scholarly journals Light-Sheet Microscopy for Surface Topography Measurements and Quantitative Analysis

Sensors ◽  
2020 ◽  
Vol 20 (10) ◽  
pp. 2842 ◽  
Author(s):  
Zhanpeng Xu ◽  
Erik Forsberg ◽  
Yang Guo ◽  
Fuhong Cai ◽  
Sailing He
Keyword(s):  
Surface Topography ◽  
Spatial Resolution ◽  
Scattered Light ◽  
Three Dimensional ◽  
Light Sheet ◽  
Field Of View ◽  
Large Field ◽  
Linear Calibration ◽  
Industrial Sample ◽  
Light Sheet Microscopy

A novel light-sheet microscopy (LSM) system that uses the laser triangulation method to quantitatively calculate and analyze the surface topography of opaque samples is discussed. A spatial resolution of at least 10 μm in z-direction, 10 μm in x-direction and 25 μm in y-direction with a large field-of-view (FOV) is achieved. A set of sample measurements that verify the system′s functionality in various applications are presented. The system has a simple mechanical structure, such that the spatial resolution is easily improved by replacement of the objective, and a linear calibration formula, which enables convenient system calibration. As implemented, the system has strong potential for, e.g., industrial sample line inspections, however, since the method utilizes reflected/scattered light, it also has the potential for three-dimensional analysis of translucent and layered structures.

scholarly journals Characterization of Scattering Effects in Phantom Samples using Single and Two-Photon Excitation Light Sheet Microscopy

2012 ◽  
Vol 102 (3) ◽  
pp. 195a-196a
Author(s):  
Zeno Lavagnino ◽  
Francesca Cella Zanacchi ◽  
Emiliano Ronzitti ◽  
Ivan Coto Hernandez ◽  
Alberto Diaspro
Keyword(s):  
Light Sheet ◽  
Excitation Light ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
Scattering Effects ◽  
Two Photon Excitation

scholarly journals Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy

2020 ◽  
Vol 11 (8) ◽  
pp. 4651
Author(s):  
Giuseppe de Vito ◽  
Pietro Ricci ◽  
Lapo Turrini ◽  
Vladislav Gavryusev ◽  
Caroline Müllenbroich ◽  
...  
Keyword(s):  
Fluorescence Emission ◽  
Light Sheet ◽  
Light Polarization ◽  
Excitation Light ◽  
Two Photon ◽  
Light Sheet Microscopy

Enhancement of image quality in planar Airy light-sheet microscopy via subtraction method

2021 ◽  
Author(s):  
Suhui Deng ◽  
Liusong Yuan ◽  
Peiwei Cheng ◽  
Yuhao Wang ◽  
Mingping Liu
Keyword(s):  
Image Quality ◽  
Light Sheet ◽  
Large Field ◽  
Subtraction Method ◽  
Optical Sectioning ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Airy Beam ◽  
Photon Excitation ◽  
Distribution Uniformity

Abstract The use of propagation-invariant Airy beams enables a light-sheet microscopy with a large field-of-view. Without relying upon two-photon excitation or deconvolution-based processing to eliminate out-of focus blur caused by the side lobes, here, we present how the subtraction method is applied to enhance the image quality in digital scanned light-sheet microscopy with Airy beam. In the proposed method, planar Airy beam with the symmetric transversal structure is used to excite the sample. A hollow Airy beam with zero intensity at the focal plane is created, which is mainly used to excite the out-of-focus signal. By scanning the sample twice with the normal planar Airy beam and the hollow Airy beam, digital post-processing of the obtained images by subtraction allows for the rejection of out-of-focus blur and improves the optical sectioning, the axial resolution and the intensity distribution uniformity of the light-sheet microscopy.

scholarly journals High-efficiency, large-area lattice light-sheet generation by dielectric metasurfaces

Nanophotonics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 4043-4051
Author(s):  
Fenghua Shi ◽  
Jing Wen ◽  
Dangyuan Lei
Keyword(s):  
Spatial Resolution ◽  
Temporal Resolution ◽  
High Efficiency ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Field Of View ◽  
Objective Lens ◽  
High Temporal Resolution ◽  
Light Sheet Microscopy ◽  
Illumination Efficiency

AbstractLattice light-sheet microscopy (LLSM) was developed for long-term live-cell imaging with ultra-fine three-dimensional (3D) spatial resolution, high temporal resolution, and low photo-toxicity by illuminating the sample with a thin lattice-like light-sheet. Currently available schemes for generating thin lattice light-sheets often require complex optical designs. Meanwhile, limited by the bulky objective lens and optical components, the light throughput of existing LLSM systems is rather low. To circumvent the above problems, we utilize a dielectric metasurface of a single footprint to replace the conventional illumination modules used in the conventional LLSM and generate a lattice light-sheet with a ~3-fold broader illumination area and a significantly leveraged illumination efficiency, which consequently leads to a larger field of view with a higher temporal resolution at no extra cost of the spatial resolution. We demonstrate that the metasurface can manipulate spatial frequencies of an input laser beam in orthogonal directions independently to break the trade-off between the field of view and illumination efficiency of the lattice light-sheet. Compared to the conventional LLSM, our metasurface module serving as an ultra-compact illumination component for LLSM at an ease will potentially enable a finer spatial resolution with a larger numerical-aperture detection objective lens.

scholarly journals A Versatile Oblique Plane Microscope for Large-Scale and High-Resolution Imaging of Subcellular Dynamics

2020 ◽  
Author(s):  
Etai Sapoznik ◽  
Bo-Jui Chang ◽  
Jaewon Huh ◽  
Robert J. Ju ◽  
Evgenia V. Azarova ◽  
...  
Keyword(s):  
High Resolution ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Membrane Dynamics ◽  
Field Of View ◽  
Large Field ◽  
Endoplasmic Reticulum Membrane ◽  
High Resolution Imaging ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

AbstractWe present an Oblique Plane Microscope that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of Lattice Light-Sheet Microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

scholarly journals Large-field high-resolution two-photon digital scanned light-sheet microscopy

Cell Research ◽  
2014 ◽  
Vol 25 (2) ◽  
pp. 254-257 ◽  
Author(s):  
Weijian Zong ◽  
Jia Zhao ◽  
Xuanyang Chen ◽  
Yuan Lin ◽  
Huixia Ren ◽  
...  
Keyword(s):  
High Resolution ◽  
Light Sheet ◽  
Large Field ◽  
Two Photon ◽  
Light Sheet Microscopy

scholarly journals A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Etai Sapoznik ◽  
Bo-Jui Chang ◽  
Jaewon Huh ◽  
Robert J Ju ◽  
Evgenia V Azarova ◽  
...  
Keyword(s):  
High Resolution ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Membrane Dynamics ◽  
Field Of View ◽  
Large Field ◽  
Endoplasmic Reticulum Membrane ◽  
High Resolution Imaging ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

scholarly journals Three-dimensional quantification of twisting in the Arabidopsis petiole

2021 ◽  
Author(s):  
Yuta Otsuka ◽  
Hirokazu Tsukaya
Keyword(s):  
Cross Sections ◽  
Three Dimensional ◽  
Light Sheet ◽  
Underlying Mechanism ◽  
Two Dimensions ◽  
Observation Angle ◽  
Differential Growth ◽  
Light Sheet Microscopy ◽  
Definition Of ◽  
Twisting Angle

AbstractOrganisms have a variety of three-dimensional (3D) structures that change over time. These changes include twisting, which is 3D deformation that cannot happen in two dimensions. Twisting is linked to important adaptive functions of organs, such as adjusting the orientation of leaves and flowers in plants to align with environmental stimuli (e.g. light, gravity). Despite its importance, the underlying mechanism for twisting remains to be determined, partly because there is no rigorous method for quantifying the twisting of plant organs. Conventional studies have relied on approximate measurements of the twisting angle in 2D, with arbitrary choices of observation angle. Here, we present the first rigorous quantification of the 3D twisting angles of Arabidopsis petioles based on light sheet microscopy. Mathematical separation of bending and twisting with strict definition of petiole cross-sections were implemented; differences in the spatial distribution of bending and twisting were detected via the quantification of angles along the petiole. Based on the measured values, we discuss that minute degrees of differential growth can result in pronounced twisting in petioles.

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