scholarly journals Automated screening by 3D light-sheet microscopy with high spatial and temporal resolution reveals mitotic phenotypes

2020 ◽  
Author(s):  
Björn Eismann ◽  
Teresa G Krieger ◽  
Jürgen Beneke ◽  
Ruben Bulkescher ◽  
Lukas Adam ◽  
...  
Keyword(s):  
Cell Cultures ◽  
High Throughput ◽  
Light Exposure ◽  
Epigenetic Modification ◽  
Fluorescent Microscopy ◽  
Light Sheet ◽  
Drug Candidate ◽  
Light Sheet Microscopy ◽  
Phenotype Classification ◽  
3D Cell Cultures

Abstract3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, three-dimensional cell spheroids are imaged for 24 hours in toto with a dual inverted selective plane illumination (diSPIM) microscope with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network based phenotype classification. We illustrate the potential of our approach by siRNA knock-down and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.

scholarly journals High-throughput live imaging using Light Sheet Microscopy

2020 ◽  
Author(s):  
Emilio J. Gualda ◽  
Matteo Bernardello ◽  
Maria Marsal ◽  
Pablo Loza Alvarez
Keyword(s):  
High Throughput ◽  
Light Sheet ◽  
Live Imaging ◽  
Light Sheet Microscopy

scholarly journals Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

2015 ◽  
Vol 9 ◽  
Author(s):  
Ludovico Silvestri ◽  
Marco Paciscopi ◽  
Paolo Soda ◽  
Filippo Biamonte ◽  
Giulio Iannello ◽  
...  
Keyword(s):  
Image Analysis ◽  
Purkinje Cells ◽  
High Throughput ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Automated High‐Throughput Drug Discovery in Peptide Hydrogel‐Based 3D Cell Cultures

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Sigrid A Langhans ◽  
Peter Worthington ◽  
Kathleen Drake ◽  
Zhiqin Li ◽  
Andrew Napper ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Cell Cultures ◽  
High Throughput ◽  
3D Cell Cultures ◽  
Peptide Hydrogel

scholarly journals A hybrid open-top light-sheet microscope for multi-scale imaging of cleared tissues

2021 ◽  
Author(s):  
Adam Glaser ◽  
Kevin Bishop ◽  
Lindsey Barner ◽  
Etsuo Susaki ◽  
Shimpei Kubota ◽  
...  
Keyword(s):  
Hybrid System ◽  
High Throughput ◽  
Light Sheet ◽  
Refractive Indices ◽  
Volumetric Imaging ◽  
Tissue Clearing ◽  
Sample Geometry ◽  
Multi Scale ◽  
Light Sheet Microscopy ◽  
User Friendly

Abstract Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a user-friendly system that can address imaging applications with varied requirements in terms of resolution (mesoscopic to sub-micrometer), sample geometry (size, shape, and number), and compatibility with tissue-clearing protocols and sample holders of various refractive indices. We present a ‘hybrid’ system that combines a novel non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet architecture for versatile multi-scale volumetric imaging.

scholarly journals Epithelial morphogenesis in the perinatal mouse uterus

2020 ◽  
Author(s):  
Zer Vue ◽  
Richard R. Behringer
Keyword(s):  
Morphological Changes ◽  
Post Partum ◽  
Fluorescent Microscopy ◽  
Light Sheet ◽  
Uterine Epithelium ◽  
List Type ◽  
Mouse Uterus ◽  
Light Sheet Microscopy ◽  
Uterine Gland ◽  
Uterine Glands

AbstractBackgroundThe uterus is the location where multiple events occur that are required for the start of new life in mammals. The adult uterus contains endometrial or uterine glands that are essential for female fertility. In the mouse, uterine glands are located in the lateral and anti-mesometrial regions of the uterine horn. Previous 3D-imaging of the adult uterus, its glands, and implanting embryos has been performed by multiple groups, using fluorescent microscopy. Adenogenesis, the formation of uterine glands, initiates after birth. Recently, we created a 3D-staging system of mouse uterine gland development at postnatal time points, using light sheet fluorescent microscopy. Here, using a similar approach, we examine the morphological changes in the epithelium of the perinatal mouse uterus.ResultsThe uterine epithelium exhibits mesometrial-antimesometrial (dorsoventral) patterning as early as three days after birth (P3), marked by the presence of the mesometrially-positioned developing uterine rail. Uterine gland buds are present beginning at P4. Novel morphological epithelial structures, including a ventral ridge and uterine segments were identified.ConclusionsThe perinatal mouse uterine luminal epithelium develops mesometrial-antimesometrial (dorsal-ventral) morphologies at 3-4 days post-partum. Between 5-6 days post-partum uterine epithelial folds form, defining alternating left-right segments.Bullet pointsMorphological patterning events in the perinatal uterine epithelium are not well described.Light sheet microscopy was used to generate volumetric reconstructions of the perinatal mouse uterine epithelium.At postnatal day 3 (P3), the uterine epithelium shows the first signs of dorsoventral pattern, with the presence of the forming mesometrially-positioned uterine rail.The first morphological indication of uterine adenogenesis begins at P4.Novel morphological structures were identified from volumetric reconstructions, including the presence of a ventral ridge (another sign of dorsoventral pattern) and uterine segmentation.

scholarly journals Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

2020 ◽  
Author(s):  
Robert H. Utama ◽  
Lakmali Atapattu ◽  
Aidan P. O’Mahony ◽  
Christopher M. Fife ◽  
Jongho Baek ◽  
...  
Keyword(s):  
Cell Cultures ◽  
High Throughput ◽  
Cell Number ◽  
Drug Dose ◽  
3D Bioprinting ◽  
Multicellular Spheroids ◽  
Drop On Demand ◽  
3D Cell Cultures ◽  
The Impact

Abstract3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of multicellular spheroids in a throughput and highly controlled manner to achieve robust and statistically relevant data, remains challenging. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of high-throughput printing of 96-well plates of spheroids. 3D-multicellular spheroids are embedded inside a tissue-like matrix with precise control over size and cell number. Application of 3D bioprinting for high-throughput drug screening was demonstrated with doxorubicin. Measurements showed that IC50 values were sensitive to spheroid size, embedding and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust high-throughput platform to screen biological and therapeutic parameters.Significance StatementIn vitro 3D cell cultures serve as more realistic models, compared to 2D cell culture, for understanding diverse biology and for drug discovery. Preparing 3D cell cultures with defined parameters is challenging, with significant failure rates when embedding 3D multicellular spheroids into extracellular mimics. Here, we report a new 3D bioprinter we developed in conjunction with bioinks to allow 3D-multicellular spheroids to be produced in a high-throughput manner. High-throughput production of embedded multicellular spheroids allowed entire drug-dose responses to be performed in 96-well plate format with statistically relevant numbers of data points. We have deconvoluted important parameters in drug responses including the impact of spheroid size and embedding in an extracellular matrix mimic on IC50 values.

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

2019 ◽  
Vol 1 (01) ◽  
pp. 1 ◽  
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

scholarly journals Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains

2015 ◽  
Vol 2 (4) ◽  
pp. 041404 ◽  
Author(s):  
M. Caroline Müllenbroich ◽  
Ludovico Silvestri ◽  
Leonardo Onofri ◽  
Irene Costantini ◽  
Marcel van’t Hoff ◽  
...  
Keyword(s):  
Data Management ◽  
High Throughput ◽  
Light Sheet ◽  
Light Sheet Microscopy
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