Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view

Light sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

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High-Resolution, Large Field-of-View, and Multi-View Single Objective Light-Sheet Microscopy

10.1101/2020.09.22.309229 ◽

2020 ◽

Author(s):

Bin Yang ◽

Alfred Millett-Sikking ◽

Merlin Lange ◽

Ahmet Can Solak ◽

Hirofumi Kobayashi ◽

...

Keyword(s):

High Resolution ◽

Light Sheet ◽

Field Of View ◽

Large Field ◽

Sample Rotation ◽

Light Sheet Microscopy ◽

Spatio Temporal ◽

Large Living ◽

Single Objective ◽

Imaging Speed

Light-sheet microscopy has become the preferred method for long-term imaging of large living samples because of its low photo-invasiveness and good optical sectioning capabilities. Unfortunately, refraction and scattering often pose obstacles to light-sheet propagation and limit imaging depth. This is typically addressed by imaging multiple complementary views to obtain high and uniform image quality throughout the sample. However, multi-view imaging often requires complex multi-objective configurations that complicate sample mounting, or sample rotation that decreases imaging speed. Recent developments in single-objective light-sheet microscopy have shown that it is possible to achieve high spatio-temporal resolution with a single objective for both illumination and detection. Here we describe a single-objective light-sheet microscope that achieves: (i) high-resolution and large field-of-view imaging via a custom remote focusing objective; (ii) simpler design and ergonomics by remote placement of coverslips; (iii) fast volumetric imaging by means of light-sheet stabilised stage scanning – a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iv) multi-view imaging by means of dual orthogonal light-sheet illumination. Finally, we demonstrate the speed, field of view and resolution of our novel instrument by imaging zebrafish tail development.

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Multiview tiling light sheet microscopy for 3D high resolution live imaging

10.1101/2021.04.18.440322 ◽

2021 ◽

Author(s):

Mostafa Aakhte ◽

Hans-Arno J Mueller

Keyword(s):

High Resolution ◽

Myosin Ii ◽

Resolution Enhancement ◽

Light Sheet ◽

Field Of View ◽

Large Field ◽

Axial Resolution ◽

Light Sheet Microscopy ◽

Drosophila Embryogenesis ◽

Mechanical Rotation

Light sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

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Light-Sheet Microscopy for Surface Topography Measurements and Quantitative Analysis

Sensors ◽

10.3390/s20102842 ◽

2020 ◽

Vol 20 (10) ◽

pp. 2842 ◽

Cited By ~ 1

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.

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

10.1101/2020.04.07.030569 ◽

2020 ◽

Cited By ~ 5

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.

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Light-sheet microscopy with isotropic, sub-micron resolution and solvent-independent large-scale imaging

10.1101/605493 ◽

2019 ◽

Cited By ~ 2

Author(s):

Tonmoy Chakraborty ◽

Meghan Driscoll ◽

Malea Murphy ◽

Philippe Roudot ◽

Bo-Jui Chang ◽

...

Keyword(s):

Dendritic Spines ◽

Large Scale ◽

Light Sheet ◽

Automated Detection ◽

Field Of View ◽

Large Field ◽

Axial Resolution ◽

Optical Sectioning ◽

Isotropic Resolution ◽

Light Sheet Microscopy

AbstractWe present cleared tissue Axially Swept Light-Sheet Microscopy (ctASLM), which achieves sub-micron isotropic resolution, high optical sectioning capability, and large field of view imaging (870×870 μm2) over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and organic chemically cleared tissue preparations and provides 2- to 5-fold better axial resolution than confocal or other reported cleared tissue light-sheet microscopes. We image millimeter-sized tissues with sub-micron 3D resolution, which enabled us to perform automated detection of cells and subcellular features such as dendritic spines.

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

eLife ◽

10.7554/elife.57681 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

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.

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Sub-voxel light-sheet microscopy for high-resolution, high-throughput volumetric imaging of large biomedical specimens

10.1101/255695 ◽

2018 ◽

Cited By ~ 1

Author(s):

Peng Fei ◽

Jun Nie ◽

Juhyun Lee ◽

Yichen Ding ◽

Shuoran Li ◽

...

Keyword(s):

High Resolution ◽

High Throughput ◽

Light Sheet ◽

Large Field ◽

Mouse Heart ◽

Processing Unit ◽

Intact Mouse ◽

Volumetric Imaging ◽

Light Sheet Microscopy ◽

Wide Range

A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). Here, we report a sub-voxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular-resolution. A non-axial, continuous scanning strategy is used to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then by adopting a sub-voxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and iteratively recovers a 3-D image with improved resolution throughout the sample. This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times, with high acquisition speeds of gigavoxels per minute. As demonstrated by quick reconstruction (minutes to hours) of various samples, e.g., 3-D cultured cells, an intact mouse heart, mouse brain, and live zebrafish embryo, the SLSM method presents a high-throughput way to circumvent the tradeoff between intoto mapping of large-scale tissue (>100 mm3) and isotropic imaging of single-cell (~1-μm resolution). It also eliminates the need of complicated mechanical stitching or precisely modulated illumination, using a simple light-sheet setup and fast graphics-processing-unit (GPU)-based computation to achieve high-throughput, high-resolution 3-D microscopy, which could be tailored for a wide range of biomedical applications in pathology, histology, neuroscience, etc.

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

10.1101/461335 ◽

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.

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Lattice light-sheet microscopy with incoherent detection and extended field of view

10.1117/12.2615734 ◽

2021 ◽

Author(s):

Mariana C. Potcoava ◽

Christopher J. Mann ◽

Simon T. Alford ◽

Jonathan Art

Keyword(s):

Light Sheet ◽

Field Of View ◽

Light Sheet Microscopy ◽

Extended Field ◽

Extended Field Of View

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