Fast confocal fluorescence imaging in freely behaving mice

Abstract

Fluorescence imaging in the brain of freely behaving mice is challenging due to severe miniaturization constraints. In particular, the ability to image a large field of view at high temporal resolution and with efficient out-of-focus background rejection still raises technical difficulties. Here, we present a novel fiberscope system that provides fast (up to 200 Hz) background-free fluorescence imaging in freely behaving mice over a field of view of diameter 230 μm. The fiberscope is composed of a custom-made multipoint-scanning confocal microscope coupled to the animal with an image guide and a micro-objective. By simultaneously registering a multipoint-scanning confocal image and a conventional widefield image, we subtracted the residual out-of-focus background and provided a background-free confocal image. Illumination and detection pinholes were created using a digital micromirror device, providing high adaptability to the sample structure and imaging conditions. Using this novel imaging tool, we demonstrated fast fluorescence imaging of microvasculature up to 120 μm deep in the mouse cortex, with an out-of-focus background reduced by two orders of magnitude compared with widefield microscopy. Taking advantage of the high acquisition rate (200 Hz), we measured red blood cell velocity in the cortical microvasculature and showed an increase in awake, unrestrained mice compared with anaesthetized animals.

Advances of optical miniscopes for in vivo imaging of neural activity in freely moving animals

“…Benefiting from the parallel recording characteristics of wide-field imaging, single-photon fluorescence miniscope has the advantage of fast imaging speeds, with typical frame rates of tens of frames per second (fps) using CMOS on headpieces ( Ghosh et al, 2011 ; Scott et al, 2018 ; Skocek et al, 2018 ; Aharoni and Hoogland, 2019 ; Liberti et al, 2022 ; Supekar et al, 2022 ), or several hundred of fps using fiber bundle to transfer images to record remotely ( Ferezou et al, 2006 ; Flusberg et al, 2008 ; Dussaux et al, 2018 ). This enables the study of bio-dynamics over a large FOV, typically with a diameter of about several hundred microns to a few millimeters.…”

Imaging the brain in action: a motorized optical rotary joint for wide field fibroscopy in freely moving animals

“…However, these are gained at the cost of a spatial discretization of the optical signal, which depends on the number of individual fibers present in the bundle. As with miniature microscopes, this is a rapidly evolving field, which has proven its potential for optical imaging of neuronal activity both at the mesoscopic scale in the cerebral cortex, 30 at the cellular scale in subcortical structures, 31,32 and for multisite photometry 33,34 in freely moving mice. The fibroscopic approach has also been used successfully to apply patterned photoactivation at the cellular scale in freely moving mice.…”

High-resolution focus-tunable one-photon miniaturized fluorescence microscope for imaging in freely moving animals

“…Since the inception of the first highly integrated mini-scope in 2011 13 , developers have continuously optimized and improved systems in order to meet growing demands of biological research. Enhancements have been made in optical performance and functional expansion, such as higher spatial resolution 14 , faster data acquisition 15 , wireless transmission 16 , multi-modality 17,18 , etc. Recent progresses in multi-photon mini-scopes provide great significance in terms of resolution, penetration depth and optical sectioning 19,20 .…”