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

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

Journal of Optics ◽

10.1088/2040-8986/ac4437 ◽

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.

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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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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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Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

Molecular Autism ◽

10.1186/s13229-021-00413-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dwaipayan Adhya ◽

George Chennell ◽

James A. Crowe ◽

Eva P. Valencia-Alarcón ◽

James Seyforth ◽

...

Keyword(s):

High Resolution ◽

Human Brain ◽

Light Sheet ◽

Autism Spectrum ◽

Model Systems ◽

Large Field ◽

Autism Spectrum Conditions ◽

Patient Specific ◽

Airy Beam

Abstract Background The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. Results Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. Conclusion We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

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