Deep two-photon brain imaging with a red-shifted fluorometric Ca ²⁺ indicator

Abstract

In vivo Ca 2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca 2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Catransients were discernable in most neurons with a good signalto-noise ratio. Action potential-dependent Ca 2+ transients were recorded in neurons of all six layers of the cortex at depths of up to −900 μm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca 2+ indicators. Ca 2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca 2+ indicator Cal-590 is well suited for in vivo twophoton Ca 2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca 2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.calcium imaging | neuronal activity | mouse cortical circuits | multicolor functional imaging

Technologies for imaging neural activity in large volumes

“…For example, for neocortex labeled with red dyes in vivo , the characteristic attenuation lengths were found to be 131 µm at 775 nm, 285 µm at 1,280 nm, and 365 µm at 1,675 nm, respectively 39,130 . Using a red-emitting calcium indicator Cal-590 and 1,050 nm excitation, Tischbirek et al recorded action potential-dependent calcium transients in all six layers of mouse cortex up to 900 µm below the pia surface in vivo 131 . Recently developed red genetically encoded calcium indicators also allowed orientation tuning properties of L6 neurons at 850 µm under pia in mouse visual cortex to be characterized in vivo 98 .…”

Advances in Two-Photon Scanning and Scanless Microscopy Technologies for Functional Neural Circuit Imaging

“…26–28]. However, greater SNR has been achieved by two new synthetic dyes, Cal-520 and Cal-590 [29, 30], which are able to reliably detect single action potentials in both brain slice and whole brain preparations, whilst remaining relatively simple to load. Cal-520 is able to detect fourfold the number of active neurons in barrel cortex in vivo when compared to OGB (see Fig.…”

“…Cal-590 is a red-shifted synthetic dye, enabling previously inaccessible neuronal calcium signals to be assessed in all six layers of cortical depth as scattering of photons at longer wavelengths is reduced. At up to 900 μ m beneath the pial surface, imaging can be performed with Cal-590 while maintaining good signal-to-noise ratio and preserving spatiotemporal fidelity [30]. …”

“…Perfusion of membrane-impermeable dyes through an intracellular recording pipette enables single action potentials, and individual synaptic inputs, to be resolved in both in vitro and in vivo preparations [29, 30, 43–48]. Bolus loading of membrane-permeable dyes enables hundreds of neurons to be examined simultaneously [18, 29, 30, 49]. There are however several costs to this.…”

Deep tissue imaging with multiphoton fluorescence microscopy

“…Two-photon fluorescence microscopy (2PM), developed in the 1990’s [1], is the most widely adopted method for minimally invasive in vivo brain imaging due to its ability to image in three dimensions. Recent in vivo imaging of neuronal circuits [2–4] and vascular networks [5,6] in the brain with micron scale resolution has revealed significant new insight into cortical function and organization. Three-dimensional multiphoton imaging is possible because of a nonlinear dependence on excitation intensity, which confines two-photon fluorescence generation to the focal volume [7].…”