Enhanced image reconstruction of three-dimensional fluorescent assays by subtractive structured-light illumination...

We investigate improved image reconstruction of structured light illumination for high-resolution imaging of three-dimensional (3D) cell-based assays. For proof of concept, an in situ fluorescence optical detection system was built with a digital micromirror device as a spatial light modulator, for which phase and tilting angle in a grid pattern were varied to implement specific image reconstruction schemes. Subtractive reconstruction algorithms based on structured light illumination were used to acquire images of fluorescent microbeads deposited as a two-dimensional monolayer or in 3D alginate matrix. We have confirmed that an optical subtraction algorithm improves axial and lateral resolution by effectively removing out-of-focus fluorescence. The results suggest that subtractive image reconstruction can be useful for structured illumination microscopy of broad types of cell-based assays with high image resolution.