Fig. 2. Wearable microscopes with custom-compound microlenses permit multi-color imaging with a single image sensor.

a Example fluorescence image of 15 μm-diameter polystyrene beads labeled with three distinct fluorophores demonstrating three-color imaging with a single RGB image sensor and dual-band filters. Scale bar, 250 μm. b, c Individual color channels before (b) and after (c) color unmixing (Methods). Scale bars, 100 μm. d Left, example fluorescence image of a 20-μm-thick mouse spinal cord section stained for astrocytes (GFAP; green) and neurons (NeuN; red) with two distinct fluorophores (Alexa Fluor 488 and 633) demonstrating two-color imaging in vitro. Scale bar, 250 μm. Center, zoom-ins of the two indicated subregions on the left. Scale bars, 50 μm. Right, color-separated images for subregion 1. e Example fluorescence image from a time-lapse recording showing neuronal nuclei (red) and surrounding astrocytes (green) in the spinal dorsal horn of a behaving mouse demonstrating multiplex imaging in vivo. Imaging was performed ~2.5 weeks after AAV9-CaMKII-H2B-GCaMP7f-TagRFP injection into the lumbar spinal cord of GFAP-GCaMP6f mice. Scale bar, 100 μm. f Color-separated images with neuronal (red) and astrocyte (green) regions of interest (ROIs) indicated. Scale bars, 100 μm. g Noxious tail pinch-evoked neuronal nuclear and astrocyte calcium transients in the ROIs shown in f (Supplementary Fig. 5; Supplementary Movie 2). The corresponding pressure stimulus and locomotor activity are shown above the activity heat maps. Running speed was recorded by placing the animal on a spherical treadmill. The vertical dashed line indicates pinch onset. All multi-color data were acquired simultaneously. All images are representatives from one sample. Images with similar properties were obtained across multiple independent samples.