Compact, easy-to-use adaptive optics module for light-sheet structural and functional imaging of neuronal structures

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.

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Light sheet microscopy for fast functional imaging of 3D neuronal cultures in hydrogels

Emerging Topics in Artificial Intelligence 2020 ◽

10.1117/12.2567263 ◽

2020 ◽

Author(s):

Gustavo Castro-Olvera ◽

Jorge Madrid-Wolff ◽

Omar E. Olarte ◽

Estefanía Estévez-Priego ◽

Adriaan A. Ludl ◽

...

Keyword(s):

Functional Imaging ◽

Light Sheet ◽

Neuronal Cultures ◽

Light Sheet Microscopy

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Whole-brain calcium imaging during physiological vestibular stimulation in larval zebrafish

10.1101/300350 ◽

2018 ◽

Author(s):

Geoffrey Migault ◽

Thomas Panier ◽

Raphaël Candelier ◽

Georges Debrégeas ◽

Volker Bormuth

Keyword(s):

Virtual Environments ◽

Functional Imaging ◽

Light Sheet ◽

Vestibular Stimulation ◽

Brain Response ◽

Whole Brain ◽

Larval Zebrafish ◽

In Vivo Functional Imaging ◽

First Time

AbstractDuring in vivo functional imaging, animals are head-fixed and thus deprived from vestibular inputs, which severely hampers the design of naturalistic virtual environments. To overcome this limitation, we developed a miniaturized ultra-stable light-sheet microscope that can be dynamically rotated during imaging along with a head-restrained zebrafish larva. We demonstrate that this system enables whole-brain functional imaging at single-cell resolution under controlled vestibular stimulation. We recorded for the first time the dynamic whole-brain response of a vertebrate to physiological vestibular stimulation. This development largely expands the potential of virtual-reality systems to explore complex multisensory-motor integration in 3D.

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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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Sensorless and sensor based adaptive optics for light sheet microscopy

Adaptive Optics and Wavefront Control for Biological Systems VI ◽

10.1117/12.2543251 ◽

2020 ◽

Author(s):

Yang Liu ◽

Keelan Lawrence ◽

James Lauderdale ◽

Peter Kner

Keyword(s):

Adaptive Optics ◽

Light Sheet ◽

Light Sheet Microscopy

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Adaptive optics light-sheet microscopy for functional neuroimaging

10.1117/12.2615699 ◽

2021 ◽

Author(s):

Antoine Hubert ◽

Fabrice Harms ◽

Sophie Imperato ◽

Vincent Loriette ◽

Cynthia Veilly ◽

...

Keyword(s):

Adaptive Optics ◽

Functional Neuroimaging ◽

Light Sheet ◽

Light Sheet Microscopy

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Highly Sensitive Shack–Hartmann Wavefront Sensor: Application to Non-Transparent Tissue Mimic Imaging with Adaptive Light-Sheet Fluorescence Microscopy

Methods and Protocols ◽

10.3390/mps2030059 ◽

2019 ◽

Vol 2 (3) ◽

pp. 59 ◽

Cited By ~ 2

Author(s):

Brajones ◽

Clouvel ◽

Dovillaire ◽

Levecq ◽

Lorenzo

Keyword(s):

Adaptive Optics ◽

Three Dimensional ◽

High Sensitivity ◽

Light Sheet ◽

Wavefront Sensor ◽

Spatial Frequencies ◽

Living Tissues ◽

Guide Star ◽

Two Photon Fluorescence ◽

Subcellular Resolution

High-quality in-depth imaging of three-dimensional samples remains a major challenge in modern microscopy. Selective plane illumination microscopy (SPIM) is a widely used technique that enables imaging of living tissues with subcellular resolution. However, scattering, absorption, and optical aberrations limit the depth at which useful imaging can be done. Adaptive optics (AOs) is a method capable of measuring and correcting aberrations in different kinds of fluorescence microscopes, thereby improving the performance of the optical system. We have incorporated a wavefront sensor adaptive optics scheme to SPIM (WAOSPIM) to correct aberrations induced by optically-thick samples, such as multi-cellular tumor spheroids (MCTS). Two-photon fluorescence provides us with a tool to produce a weak non-linear guide star (NGS) in any region of the field of view. The faintness of NGS; however, led us to develop a high-sensitivity Shack–Hartmann wavefront sensor (SHWS). This paper describes this newly developed SHWS and shows the correction capabilities of WAOSPIM using NGS in thick, inhomogeneous samples like MCTS. We report improvements of up to 79% for spatial frequencies corresponding to cellular and subcellular size features.

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