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Fast, small and many! How confocal calcium imaging can be achieved from thousands of sites at high temporal resolution

20 Jun | By Biophotonics.World
Fast, small and many! How confocal calcium imaging can be achieved from thousands of sites at high temporal resolution
Confocal image of a cerebellar Purkinje neuron filled with the Ca2+ indicator Oregon Green BAPTA-5N. The fluorescence peak signal associated with stimulated parallel fibre synaptic potential is reported in superimposed colour scale. The time course of the change in fluorescence (ΔF/F0) in three submicron sites is reported on the left.
Image source: Marco Canepari
By: Marco Canepari

The team of Marco Canepari, from the Laboratory of Interdisciplinary Physics in Grenoble (https://www-liphy.ujf-grenoble.fr/), in collaboration with the companies Cairn Research (https://www.cairn-research.co.uk/), Scimeasure (https://www.scimeasure.com/) and Crest Optics (http://www.crestopt.com/), recently developed a rapid confocal imaging system based on a fast spinning disk and a sensitive CMOS camera. The system was designed to perform functional imaging, in particular calcium imaging, from ex-vivo tissues such as brain slices. The system is described in one of the latest issues of Journal of Biophotonics.

Confocal microscopy is widely used in biology to improve the spatial discrimination of sub-micron structures and to achieve optical sectioning in 3D tissues. The intrinsic requirement of resolving a change in fluorescence from the photon noise, however, poses important limitations when confocal imaging is performed using the conventional scanning approach, i.e. by raster scanning a focussed laser spot over the sample using a single pinhole and point detector. Indeed, at a desired fixed acquisition frequency, the exposure time and therefore the number of photons at each point decrease with the number of points. Further to this the instantaneous localised power required is damaging to living tissue and the serial nature of the acquisition limits the effective frame rates. To increase acquisition rates, reduce phototoxicity and increase signal beyond the noise floor a parallel imaging approach is needed, typically, a spinning Nipkow disk in combination with a camera to multiplex the scans.

While the conceptual approach of confocal imaging using a spinning disk has been utilised for many years, novel technology was required to develop an optimised system specifically targeted to investigation of calcium signals in submicron neuronal compartments in brain slices, in combination with patch clamp recordings.This new system assembles an optimised fast spinning disk, a multimode diode laser and a novel high-resolution CMOS camera. The spinning disk, running at 20,000 rpm, has a custom-designed spiral pattern maximising light collection and rejecting out-of-focus fluorescence to discriminate signals from small neuronal compartments.

Using a 60X objective, it was possible to record from tens of thousands of pixels at the resolution of ~250 nm per pixel in the kHz range with 14-bit digitisation. It was possible to resolve physiological Ca2+ transients from submicron structures at 20-40 µm below the slice surface, using a low affinity calcium indicator to preserve the native kinetics of the signal. Signals up to 1.25 kHz were resolved in single trials, or through averages of only 2-8 recordings, from dendritic spines and small parent dendrites in neurons. In these experimental conditions, the system performs optimally whereas wide-field illumination fails to discriminate signals with submicron resolution, in particular because it lacks the necessary axial sectioning.

The understanding of fast calcium signaling in small axonal or dendritic compartments and in synaptic spines is fundamental in neurobiology. Although a final goal in biophotonics applied to neurosciences is to perform functional imaging in the intact brain, the acute brain slice is a critical preparation since it permits accessing native neurons easily and with only a thin layer of tissue between the objective and the recording cell. This new confocal system, which is now commercially available through Cairn Research, has been conceived to be ideal for this purpose. It can be easily coupled to commercial microscopes and combined with electrophysiology; it does not require specific knowledge in optics; it is affordable and it requires minimal maintenance. Thus, the system offers an interesting economic solution for functional imaging in brain slices.

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