Fig. 1: Illustrations of full-color HiLo microscopy.

a Schematic of full-color HiLo microscopy. Two shots are required for each FOV. Under speckle illumination (left), the beam from the 532 nm laser diode is projected to the surface of the diffuser. The resulting speckle pattern is relayed to the back focal plane of the objective lens with a pair of expansion lenses, and finally projected onto the surface of the imaged sample. The excited fluorescence signals will then be collected by the objective lens, pass through the beam splitter, and finally be focused on the color camera. Under uniform illumination (right), the LD is powered off, and two UV-LEDs are activated, projecting UV beams obliquely from two slides. The fluorescence signals will follow the same collection path as in the speckle image acquisition step. b–g An intuitive example illustrating the principles of full-color HiLo microscopy, where speckle illumination image (\({I}_{s}\)), uniform illumination image (\({I}_{u}\)), speckle contrast (\({C}_{s}\)), in-focus low-frequency components (\({I}_{{Lo}}\)), and the final output HiLo image (\({I}_{{HiLo}}\)) of the same FOV of a mouse kidney is presented. LD, laser diode; L1, lens 1; L2, lens 2; OL, objective lens; BS, beam splitter; NF, notch filter; TL, tube lens; M, mirror. Note: images in b–g have been normalized with min-max normalization for better visualization.