Utilizing quantitative phase microscopy to localize fluorescence in three dimensions via the transport of intensity equation.

We demonstrate the use of a novel, to the best of our knowledge, localization algorithm for digitally refocusing fluorescence images from a three-dimensional cell culture. Simultaneous phase and fluorescence intensity images are collected through a multimodal system that combines digital holography via quantitative phase microscopy (QPM) and fluorescence microscopy. Defocused fluorescence images are localized to a specific z-plane within the three-dimensional (3D) matrix using the transport of intensity equation (TIE) and depth-resolved information derived from the QPM measurements. This technique is applied to cells stained with different fluorescent tags suspended in 3D collagen hydrogel cultures. Experimental findings demonstrate the localization of defocused images, facilitating the analysis and comparison of cells within the hydrogel matrix. This method holds promise for comprehensive cellular imaging of fluorescence labeling in three-dimensional environments, enabling detailed investigations into cellular behavior and interactions.