Focal modulation microscopy: Theory and implementation

Focal modulation microscopy (FMM) is a novel method for high-resolution imaging of thick biological tissues. It is compatible with both fluorescence and scattering/reflection contrast mechanisms and has demonstrated a penetration depth significantly greater than that of confocal microscopy (CM). To predict the improved imaging performance, we have developed a theoretical model that combines wave optics and Monte Carlo method to simulate light propagation in turbid medium and imaging formation processes. It is found that FMM can provide a signal to background ratio (SBR) about 15 dB better than CM at the imaging depth of 100 microns, and the improvement increases to more than 35 dB when the focal depth reaches 500 microns. In order to take full advantage of the remarkable background rejection capability of FMM and maximize the penetration depth, a great deal of attention should be paid to the signal to noise ratio (SNR), which is limited by the shot noise. The SNR is related to the modulation depth, which depends on the design and implementation of the spatial-temporal modulator. We have evaluated a number of pupil functions that have different number of segments and configurations. When the modulation depth is improved with an increasing number of segments in general, a modulator with four to six segments seems a good compromise between signal quality and the ease of fabrication. We are also investigating post-processing algorithms that can partially remove the random noises in FMM images.