Fig. 2
From: Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy
Submicrometer-scale channel registration over millimeter-scale volumes. ChroMS provides multicolor excitation in the 850–1100 nm range with <0.6 µm of chromatic shift in all three directions in the used field of view and at arbitrary depths. a Overlap of the two PSFs is required for wavelength mixing. Axial and lateral chromatic shifts are measured using SHG nanocrystals. Top right: coalignment of the foci at the center of the field of view (fov). Bottom right: axial PSFs corresponding to the two beams measured were axially matched using telescopes placed on each beam path. b Characterization of chromatic aberrations across the fov for the 850 nm/1100 nm wavelength combination. Top: Chromatic lateral shifts ΔX and ΔY as a function of the position in the fov. Measurements were performed on n = 1012 KTP nanocrystals across the fov and data fitted using an affine model. Bottom left: Chromatic axial shift ΔZ as a function of the radial position in the fov. Measurements were performed on n = 1037 KTP nanocrystals across the fov and data fitted using a polynomial model. Bottom right: Lateral chromatic shift mapped across the fov. Lateral chromatic shift is defined as (ΔX2 + ΔX2)0.5 with ΔX and ΔY the corresponding fitted horizontal and vertical chromatic shifts. Dashed boxes outline the effective fov used for ChroMS imaging experiments. c Hue-Saturation polar plot representing the distribution of color combinations expressed in a portion of cortex labeled with the MAGIC markers strategy after spectral unmixing. Multiple color combinations (>20) spanning the HSV color space can be distinguished14. d XY representative multicolor images from a MAGIC markers-labeled dataset acquired with ChroMS microscopy with 0.4 µm × 0.4 µm × 1.5 µm voxel size. Insets demonstrate channel co-registration at arbitrary depths within the dataset. Individual channels (R, G, B) are shown in gray scale. Cell structures (astrocyte domains and main processes, cell bodies, axons) can be visualized with multicolor precision (i.e. not affected by chromatic aberrations) and discriminated with a quantitative color ratio. Scale bar: 100 µm
