Axial localization with modulated-illumination extended-depth-of-field microscopy.

High-speed volumetric imaging represents a challenge in microscopy applications. We demonstrate a technique for acquiring volumetric images based on the extended depth of field microscopy with a fast focal scan and modulated illumination. By combining two frames with different illumination ramps, we can perform local depth ranging of the sample at speeds of up to half the camera frame rate. Our technique is light efficient, provides diffraction-limited resolution, enables axial localization that is largely independent of sample size, and can be operated with any standard widefield microscope based on fluorescence or darkfield contrast as a simple add-on. We demonstrate the accuracy of axial localization and applications of the technique to various dynamic extended samples, including in-vivo mouse brain.