A team from the Institute for Neuroscience at the Technical University of Munich published a report on their experience of deep two-photon brain imaging with a red-shifted fluorometric calcium indicator. The indicator in question is Cal-590™, which emits a signal of 588nm after excitation with infrared light. Within their report, Tischbirek and his team demonstrate how Cal-590™ is suitable for deep tissue in vivo experiments in all six layers of the mouse cortex, and also show how Cal-590™ can be detected in combination with other calcium indicators in multicolor functional imaging experiments.

To prepare for the use of Cal-590™ in two-photon imaging, Tischbirek and his team adjusted their two-photon microscope for long wavelength measurement and determined the two-photon excitation spectrum of Cal-590™, finding the maximum to be around 1,050-1,060 nm. These wavelengths fall within the second near-infrared window, meaning they exhibit low levels of absorption and scattering of photons by blood, fat, and water, which identifies them as suitable for deep-imaging purposes. This excitation maximum also happens to make Cal-590™ compatible for two-photon imaging use with a subclass of ytterbium fiber lasers that emits a beam centered around 1,050 nm.

Following the bulk-loading of layer 2/3 neurons in the mouse visual cortex with Cal-590™ AM, the team noticed the somata of the dyed neurons had a ring-like appearance, which is explainable given Cal-590™'s enhanced intracellular dye retention properties. Cal 590 remains within the cytoplasm and defines the cell shape as a whole, rather than compartmentalizing into the nucleus or mitochondria as other red indicators, like Rhod-2, would. In comparing high-speed two-photon Cal-590™ recordings of neural activity with cell-attached patch-clamp recordings that were taken simultaneously, Cal-590™ was also found to be useful in adequately monitoring calcium transients associated with spike activity. Such calcium transients were distinguishable because of Cal-590™'s great signal-to-noise ratio and rapid rise and decay times. Tischbirek and his team proceeded to obtain images of layer 4 neurons with comparable signal-to-noise ratio using the same bulk-loading staining technique at depths of -450 to -520 µm.

At layer 5/6, individual neurons were filled with Cal-590™ via electroporation, using a shadow-patching approach. Tischbirek emphasizes that at such depths, restriction of staining to the layer and section of interest is fundamental to minimizing any out-of-focus fluorescence. Here is another area where using Cal-590™ is favorable in two-photon application; its exceptional dye retention qualities reduce any cell leakage that may affect image resolution. A comparison of cell-attached and dendritic two-photon calcium imaging recordings showed that the peaks of individual calcium transients were still discernible, even in the high-frequency trains of action potentials (APs) that are more commonly generated in the deeper cortical layers (approximately -740 µm). Amplitudes of calcium transients linearly reported the number of APs occurring in both dendrites and somata.

To prove that Cal-590™ was also a good dye candidate for dual-color functional calcium imaging, Tischbirek and his team prepared an image in which a population of deep layer 4 neurons were bulk-labeled with Cal-590™, and a neuron at the same layer was electroporated with a green fluorescent indicator. The resulting recordings and image had good signal-to-noise ratio with no bleed-through between the two dyes, identifying distinctive calcium transients in multiple somatic cells, and in the dendrites of the green stained neuron.

Cal-590™ has greatly improved Tischbirek's team's ability to monitor neuronal activity up to -900 µm below the pia, and was also found to be suitable for multicolor imaging. Cal-590™'s advantages over alternative two-photon imaging calcium indicators includes its versatility and convenience as a synthetic indicator dye, and its cytosolic retention and rapid kinetics contributing to high resolution in resulting data, making it especially fitted for examining highly active cells. Additionally, no photobleaching was observed during the extent of the experiments. Given these attributes, Cal-590™ has more potential to enhance other in vivo deep-imaging techniques.