Modulated Alignment Dual-Axis (MAD) Confocal Microscopy for Deep Optical Sectioining in Tissues

We present a strategy to preserve contrast over a deeper range of imaging depths in the context of confocal microscopy using low-power (0.5 mW) diode laser illumination. While the dual-axis confocal microscope architecture´s intersecting illumination and collection beams significantly improves the spatial-filtering and optical-sectioning performance of confocal microscopy, we propose that modulating the spatial alignment of the dual-axis beams at a frequency f will generate a focal volume signal modulated at 2f, which further provides nearly an order-of-magnitude improvement in optical-sectioning contrast. Lock-in detection is used to remove the unmodulated background light, thereby enhancing our ability to image deeply within highly scattering tissues.