Structured illumination enhances resolution and contrast in thick tissue fluorescence imaging

We introduce a noncontact imaging method utilizing multifrequency structured illumination for improving lateral and axial resolution and contrast of fluorescent molecular probes in thick, multiple-scattering tissue phantoms. The method can be implemented rapidly using a spatial light modulator and a simple image demodulation scheme similar to structured light microscopy in the diffraction regime. However, imaging is performed in the multiple-scattering regime utilizing spatially modulated scalar photon density waves. We demonstrate that by increasing the structured light spatial frequency, fluorescence from deeper structures is suppressed and signals from more superficial objects enhanced. By measuring the spatial frequency dependence of fluorescence, background can be reduced by localizing the signal to a buried fluorescent object. Overall, signal-to-background ratio (SBR) and resolution improvements are dependent on spatial frequency and object depth/dimension with as much as sevenfold improvement in SBR and 33% improvement in resolution for ~1-mm objects buried 3 mm below the surface in tissue-like media with fluorescent background.