A novel multisite confocal system for rapid Ca²⁺ imaging from submicron structures in brain slices

Abstract

In brain slices, resolving fast Ca fluorescence signals from submicron structures is typically achieved using 2-photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high-resolution CMOS camera. The spinning disk, running at 20 000 rpm, has custom-designed spiral pattern that maximises light collection, while rejecting out-of-focus fluorescence to resolve signals from small neuronal compartments. Using a 60× objective, the camera permits acquisitions of tens of thousands of pixels at resolutions of ~250 nm per pixel in the kHz range with 14 bits of digital depth. The system can resolve physiological Ca transients from submicron structures at 20 to 40 μm below the slice surface, using the low-affinity Ca indicator Oregon Green BAPTA-5N. In particular, signals at 0.25 to 1.25 kHz were resolved in single trials, or through averages of a few recordings, from dendritic spines and small parent dendrites in cerebellar Purkinje neurons. Thanks to an unprecedented combination of temporal and spatial resolution with relatively simple implementation, it is expected that this system will be widely adopted for multisite monitoring of Ca signals.

Optical measurement of physiological sodium currents in the axon initial segment

“…Optical recordings were done using a modified version of a previously used system (Filipis et al 2018).…”

“…1C). We have previously estimated that for a fluorescent structure at 20-30 µm from the slice surface, which is the case of an intact axon, fluorescence can be discriminated with a precision of 2-3 µm (Filipis et al 2018). Thus, to improve the signal-to-noise ratio (SNR) within the factual spatial resolution, we replaced the fluorescence F at each point ξ on the x-axe, with the Gaussian filtered fluorescence G(ξ) defined by the equation:…”

The Origin of Physiological Local mGluR1 Supralinear Ca2+Signals in Cerebellar Purkinje Neurons

“…This study was achieved by monitoring fluorescence simultaneously in large portions of PN dendrites with a spatial resolution of ϳ10 M. Thus, the dendritic sites of recording comprise several spines and the parent dendritic segment. A full understanding of the signals in terms of individual spines and dendritic bulks, which is not available in this study, should be achieved in the near future either by reducing the temporal resolution using rapid multisite confocal imaging (Filipis et al, 2018) or by reducing the number of recording spots using two-photon random access microscopy (Otsu et al, 2008). A second question that should be explored in detail is the relation between the initial V m state and the induction of PF synaptic plasticity.…”

The origin of physiological local mGluR1 supralinear Ca2+ signals in cerebellar Purkinje neurons

“…Thus, the dendritic sites of recording comprise several spines and the parent dendritic segment. A full understanding of the signals in terms of individual spines and dendritic bulks, which is not available in this study, should be achieved in the near future either by reducing the temporal resolution using rapid multisite confocal imaging (Filipis et al, 2018), or by reducing the number of recording spots using 2-photon random access microscopy (Otsu et al, 2008). A second question that should be explored in detail is the relation between the initial V m state and the induction of PF synaptic plasticity.…”