Simultaneous multicolor imaging using off-axis spectral encoding in a single camera without sacrificing frame rate

Multicolor imaging has been widely applied across various biological and medical applications, especially essential for probing diverse biological structures. However, existing multicolor imaging methods often sacrifice either simultaneity or speed, posing a challenge for simultaneous imaging of over three fluorophores. Here, we proposed off-axis spectral encoding multicolor microscopy (OSEM) with a single camera that simultaneously captures encoded multicolor signals and reconstructs monochromatic images by decoding. Based on the natural intensity modulation difference of a single illumination spot across off-axis detection positions, we adjusted the multicolor excitation beams with distinct off-axis offsets from the same detection position to achieve spectral encoding. The method achieved multicolor simultaneous imaging in a single camera without extra sacrifice of frame rate. We evaluated OSEM’s imaging performance by imaging multicolor synthetic samples and fluorescent microbeads. We also demonstrated that OSEM reduced imaging time by 5.8 times and achieved 99% accuracy in classifying and counting multicolor fluorescent bacteria, outperforming sequential imaging. We obtained four-color fluorescent optical-sectioning images of a mouse brain slice at a speed of 2.85 mm 2 /s, demonstrating its effectiveness for high-throughput multicolor imaging of large tissue samples. These results indicate that OSEM offers a reliable and efficient tool for multicolor fluorescent imaging of large biological tissues.