a Experimental diagram of the MATRIEX imaging system. The two round 3D objects in the lower-left corner are the top and bottom views of the mouse head chamber used for in vivo imaging. (Ti:Sa): Ti:Sapphire ultrafast pulsing laser; PC: Pockels cell; BE: beam expander; SM1 and SM2: x–y scanning mirrors; SL: scan lens; TL: tube lens; DM: dichroic mirror; CL: collection lens; PMT: photomultiplier tube; DO: dry objective; MOs: miniaturized objectives. b Photograph showing an oblique overview of the actual MATRIEX imaging system. c The photograph in the upper image shows a zoomed in view of the three MOs attached to the manipulating bars over the head chamber; the lower photograph was taken directly above the MOs with a smartphone camera. All MOs used in this figure are of the same model: ‘standard version’ (see Table 1). d, e Illustrations of the two-stage magnification and multiaxis coupling. The square images are actual two-photon images taken of 20-μm beads. Each red circle indicates one FOV. The model of DO used in panels d-f is the Olympus MPlan ×4/0.1, and all MOs in this figure are of the same customized model (‘Standard version’, see Table 1). f Illustration showing the absence of inter-FOV crosstalk under adjacent MOs. The images were taken on a uniform fluorescent plate. The red circles indicate the areas of analysis used to compare the image contrast between two conditions; the left-side condition shows the fluorescent plate under both MOs, and the right-side condition shows the fluorescence plate under only one MO. g Testing the optical resolution of the compound assembly with 0.51-μm beads. Curves: Gaussian fittings of raw data points. The on-axis or off-axis fluorescence intensity profiles were measured when the axis of the MO was aligned with the axis of the DO or apart from the axis of the DO (2 mm for the DO of ×4 or ×5, 3 mm for the DO of ×2.5, and 4 mm for the DO of ×2), respectively. See Table 2 for a summary of the measurements.