Bram, L.; Tal-Friedman, O.; Fibeesh, N.; Bar-Sinai, Y.; Flaxer, E.; Roichman, Y.; Ebenstein, Y.; Jeffet, J.

Fluorescence confocal microscopy is a fundamental and widely used tool for biological, medical, and chemical research. It enables color-coded visualization of sample components, enhancing our understanding of life\'s building blocks and their interactions. The recent realization of image scanning microscopy (ISM) using confocal spinning disks (CSD) enhanced the spatial resolution of fluorescence microscopy to twice the diffraction limit with minimal sample perturbation and fast acquisition rates. However, capturing multi-color images using ISM is still time-consuming and introduces temporal artifacts as different colors are not acquired simultaneously. Here, we present a spectral CSD-ISM system designed for concurrent high-resolution and simultaneous multi-color acquisition. By integrating a custom linear Amici prism into the CSD-ISM optical detection path, we achieve multi-color, super-resolution images at a fraction of the acquisition time and with a flexible color palette selection. A digital signal processor (DSP) is employed together with accompanying software as a cost-effective alternative to Field-Programmable Gate Arrays (FPGAs) used in previous studies. We provide an accompanying GPU-compatible, python-based image processing pipeline to decompose spectral signatures into multi-color channel-based images, preserving the optical resolution. System characterization using three color fluorescent beads demonstrated 1.74-fold resolution improvement over the diffraction limit and accurate color classification with three times faster acquisition compared to standard CSD-ISM. Application to neuron cells expressing a Parkinson\'s disease-associated mutation, showcased improved resolution and contrast of four distinctly labeled cellular components. This spectral CSD-ISM system provides a valuable tool for biological imaging, enabling the simultaneous acquisition of high-resolution spatial information and multi-color spectral data.