Multimodal microscopy aims at combining complementary images obtained from different imaging techniques. We have developed a custom built microscope that is capable of separate or simultaneous image acquisition from multiple optical imaging modalities, such as bright-field and fluorescence microscopy. The use of an electrically focus-tunable lens in the microscope allows us to acquire multi-focus z-stacks of thick 3D biological samples. This information can be combined by post-processing algorithms allowing for image reconstruction of "new" images with relevant information e.g., extended depth of field as well as 3D visualization of the sample. After calibration of the ETL and image registration of the z-stack, algorithms are performed in Fourier domain without segmentation of the focused regions or estimation of the depth map, which usually introduce inaccuracies into the reconstruction.
Limited depth-of-field can be overcome through computational optical imaging. In this work, a custom built fluorescence microscope with a focus electrically tunable lens is used for the acquisition of a multifocus image sequence (z-stack) of a 3D fluorescent sample. Image registration between the acquired images is often needed as a preprocessing step before the reconstruction of images with new characteristics. Then a multifocus image fusion algorithm is implemented to accomplish all-in-focus image reconstruction from the registered z-stack. Also computational perspective shifts that allow to the reconstruction of stereoscopic pairs of the sample and its three-dimensional visualization are implemented.