Engineered fluidic device to achieve multiplexed monitoring of cell cultures with digital holographic microscopy

Bélanger, Erik; Adiba, Carine Ben; Rioux-Péllerin, Émile; Becq, Frédéric; Jourdain, Pascal; Marquet, Pierre

doi:10.1364/oe.444701

Cited by 3 publications

(1 citation statement)

References 32 publications

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“…In preparation for live Ca 2+ imaging experiments, the neural culture medium was replaced with ACSF buffer and acclimated for 30 min in an incubator before imaging, ensuring optimal conditions for the imaging process. Then, CSs were transferred into a custom-designed perfusion chamber (Bélanger et al, 2019(Bélanger et al, , 2022. During the Ca 2+ imaging, the perfusion chamber and ACSF buffer temperature were maintained at 37°C while the buffer flow rate was 1 mL/min (Lévesque et al, 2018).…”

Section: Live Cell Ca 2+ Imagingmentioning

confidence: 99%

A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons

Patel,

Ouellet,

Paquet-Mercier

et al. 2023

Front. Neurosci.

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IntroductionHuman induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process.MethodsIn this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter.ResultsThe retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types.DiscussionOur results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.

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Section: Live Cell Ca 2+ Imagingmentioning

confidence: 99%

A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons

Patel,

Ouellet,

Paquet-Mercier

et al. 2023

Front. Neurosci.

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Visualizing the fine structure and dynamics of living cells with temporal polychromatic digital holographic microscopy

Haouat,

Larivière-Loiselle,

Crochetière

et al. 2024

J. Opt. Soc. Am. A

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Polychromatic digital holographic microscopy (P-DHM) has demonstrated its capacity to generate highly denoised optical path difference images, thereby enabling the label-free visualization of fine cellular structures, such as the dendritic arborization within neuronal cells in culture. So far, however, the sample must remain more or less stationary since P-DHM is performed manually, i.e., all actions are carried out sequentially over several minutes. In this paper, we propose fully automated, robust, and efficient management of the acquisition and reconstruction of the time series of polychromatic hologram sets, transforming P-DHM into temporal P-DHM. Experimental results have demonstrated the ability of the proposed temporal P-DHM implementation to non-invasively and quantitatively reveal the fine structure and dynamics of living cells.

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Focus issue introduction: 3D image acquisition and display: technology, perception and applications

Javidi¹,

Hua

Martı́nez-Corral

et al. 2022

Opt. Express

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This Feature Issue of Optics Express is organized in conjunction with the 2021 Optica (OSA) conference on 3D Image Acquisition and Display: Technology, Perception and Applications which was held virtually from 19 to 23, July 2021 as part of the Imaging and Sensing Congress 2021. This Feature Issue presents 29 articles which cover the topics and scope of the 2021 3D conference. This Introduction provides a summary of these articles.

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Engineered fluidic device to achieve multiplexed monitoring of cell cultures with digital holographic microscopy

Cited by 3 publications

References 32 publications

A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons

A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons

Visualizing the fine structure and dynamics of living cells with temporal polychromatic digital holographic microscopy

Focus issue introduction: 3D image acquisition and display: technology, perception and applications

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