An optical recording system based on a fast CCD sensor for biological imaging

Mammano, Fabio; Canepari, Marco; Capello, G.; Ijaduola, R.; Cunei, Antonio; Ying, Liliang; Fratnik, F.; Colavita, A.

doi:10.1054/ceca.1998.0013

Cited by 20 publications

(14 citation statements)

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“…Image sequences of Vf2.1.Cl fluorescence were acquired continuously at 10 frames per second with 100 ms exposure time. To synchronize image acquisition and electrical recordings, we sampled the 5 V pulse (FVAL) that signals active exposure of the CCD camera [46]. Vf2.1.Cl signals were measured as relative changes of fluorescence emission intensity (Δ F/F 0 ), where F 0 is prestimulus fluorescence, F is fluorescence at time t and Δ F = F – F 0 .…”

Section: Methodsmentioning

confidence: 99%

A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks

Ceriani

Mammano

2013

Cell Commun Signal

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BackgroundA variety of mechanisms that govern connexin channel gating and permeability regulate coupling in gap junction networks. Mutations in connexin genes have been linked to several pathologies, including cardiovascular anomalies, peripheral neuropathy, skin disorders, cataracts and deafness. Gap junction coupling and its patho–physiological alterations are commonly assayed by microinjection experiments with fluorescent tracers, which typically require several minutes to allow dye transfer to a limited number of cells. Comparable or longer time intervals are required by fluorescence recovery after photobleaching experiments. Paired electrophysiological recordings have excellent time resolution but provide extremely limited spatial information regarding network connectivity.ResultsHere, we developed a rapid and sensitive method to assay gap junction communication using a combination of single cell electrophysiology, large–scale optical recordings and a digital phase–sensitive detector to extract signals with a known frequency from Vf2.1.Cl, a novel fluorescent sensor of plasma membrane potential. Tests performed in HeLa cell cultures confirmed that suitably encoded Vf2.1.Cl signals remained confined within the network of cells visibly interconnected by fluorescently tagged gap junction channels. We used this method to visualize instantly intercellular connectivity over the whole field of view (hundreds of cells) in cochlear organotypic cultures from postnatal mice. A simple resistive network model reproduced accurately the spatial dependence of the electrical signals throughout the cellular network. Our data suggest that each pair of cochlear non−sensory cells of the lesser epithelial ridge is coupled by ~1500 gap junction channels, on average. Junctional conductance was reduced by 14% in cochlear cultures harboring the T5M mutation of connexin30, which induces a moderate hearing loss in connexin30T5M/T5M knock–in mice, and by 91% in cultures from connexin30−/− mice, which are profoundly deaf.ConclusionsOur methodology allows greater sensitivity (defined as the minimum magnitude of input signal required to produce a specified output signal having a specified signal−to−noise ratio) and better time resolution compared to classical tracer–based techniques. It permitted us to dynamically visualize intercellular connectivity down to the 10th order in non−sensory cell networks of the developing cochlea. We believe that our approach is of general interest and can be seamlessly extended to a variety of biological systems, as well as to other connexin−related disease conditions.

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Section: Methodsmentioning

confidence: 99%

A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks

Ceriani

Mammano

2013

Cell Commun Signal

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“…Single action potential detection was prevented during high frequency bursts due to the relatively long fl uorescence decay time constant of Fluo-4 (300 ± 11 ms, Mean ± S.E.) [ 12 ]. 2.…”

Section: For Detection Of Camentioning

confidence: 99%

Design and Construction of a Cost-Effective Spinning Disk System for Live Imaging of Inner Ear Tissue

Ceriani

Ciubotaru

Bortolozzi

et al. 2016

Methods in Molecular Biology

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Confocal imaging of fluorescent probes offers a powerful, non-invasive tool which enables data collection from vast population of cells at high spatial and temporal resolution. Spinning disk confocal microscopy parallelizes the imaging process permitting the study of dynamic events in populations of living cells on the millisecond time scale. Several spinning disk microscopy solutions are commercially available, however these are often poorly configurable and relatively expensive. This chapter describes a procedure to assemble a cost-effective homemade spinning disk system for fluorescence microscopy, which is highly flexible and easily configurable. We finally illustrate a reliable protocol to obtain high-quality Ca(2+) and voltage imaging data from cochlear preparations.

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“…The temporal firing pattern itself could be an additional method of information representation within a neuronal circuit, contributing to rapid temporal coding (VanRullen et al, 2005). Using multi-cellular calcium imaging, activation patterns of multiple cells (>100 neurons) can be studied and analyzed (Mammano et al, 1999; Garaschuk et al, 2000; Stosiek et al, 2003; Hirase et al, 2004; Ikegaya et al, 2004; Cossart et al, 2005b). This provides information on functional connectivity and insights into circuit dynamics (Takahashi et al, 2007; Bonifazi et al, 2009; Sasaki et al, 2009; Rothschild et al, 2010; Hofer et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Deterministic and Stochastic Neuronal Contributions to Distinct Synchronous CA3 Network Bursts

Takano

McCartney²,

Ortinski³

et al. 2012

J. Neurosci.

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Computational studies have suggested that stochastic, deterministic, and mixed processes all could be possible determinants of spontaneous, synchronous network bursts. In the present study, utilizing multicellular calcium imaging coupled with fast confocal microscopy, we describe neuronal behavior underlying spontaneous network bursts in developing rat and mouse hippocampal area CA3 networks. Two primary burst types were studied: giant depolarizing potentials (GDPs) and spontaneous interictal bursts recorded in bicuculline, a GABAA receptor antagonist. Analysis of the simultaneous behavior of multiple CA3 neurons during synchronous GDPs revealed a repeatable activation order from burst to burst. This was validated using several statistical methods, including high Kendall’s W values for firing order during GDPs, high Pearson’s correlations of cellular activation times between burst pairs, and latent class analysis, which revealed a population of 5-6% of CA3 neurons reliably firing very early during GDPs. In contrast, neuronal firing order during interictal bursts appeared homogenous, with no particular cells repeatedly leading or lagging during these synchronous events. We conclude that GDPs activate via a deterministic mechanism, with distinct, repeatable roles for subsets of neurons during burst generation, while interictal bursts appear to be stochastic events with cells assuming interchangeable roles in the generation of these events.

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An optical recording system based on a fast CCD sensor for biological imaging

Cited by 20 publications

References 0 publications

A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks

A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks

Design and Construction of a Cost-Effective Spinning Disk System for Live Imaging of Inner Ear Tissue

Deterministic and Stochastic Neuronal Contributions to Distinct Synchronous CA3 Network Bursts

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