An AutoPilot platform for high-resolution light-sheet microscopy

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

Development ◽

10.1242/dev.199725 ◽

2021 ◽

Author(s):

Mostafa Aakhte ◽

H.-Arno J. Müller

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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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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Subvoxel light-sheet microscopy for high-resolution high-throughput volumetric imaging of large biomedical specimens

Advanced Photonics ◽

10.1117/1.ap.1.1.016002 ◽

2019 ◽

Vol 1 (01) ◽

pp. 1 ◽

Cited By ~ 15

Author(s):

Peng Fei ◽

Jun Nie ◽

Juhyun Lee ◽

Yichen Ding ◽

Shuoran Li ◽

...

Keyword(s):

High Resolution ◽

High Throughput ◽

Light Sheet ◽

Volumetric Imaging ◽

Light Sheet Microscopy

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RECONSTRUCTING EMBRYONIC DEVELOPMENT FROM HIGH-RESOLUTION LIGHT SHEET MICROSCOPY DATA

Computational Modeling: From Chemistry to Materials to Biology ◽

10.1142/9789811228216_0026 ◽

2021 ◽

Author(s):

JAN HUISKEN

Keyword(s):

High Resolution ◽

Embryonic Development ◽

Light Sheet ◽

Light Sheet Microscopy ◽

Microscopy Data

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High-resolution light-sheet microscopy: a simulation of an optical illumination system for oil immersion

10.1117/12.2190823 ◽

2015 ◽

Author(s):

Xiang Lu ◽

Rainer Heintzmann ◽

Ulrich Leischner

Keyword(s):

High Resolution ◽

Light Sheet ◽

Light Sheet Microscopy ◽

Illumination System ◽

Oil Immersion

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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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