Modulated-Alignment Dual-Axis (MAD) Confocal Microscopy Optimized for Speed and Contrast

Modulated-alignment dual-axis (MAD) confocal microscopy combines the benefits of dual-axis confocal (DAC) microscopy and focal-modulation microscopy (FMM) for rejecting out-of-focus and multiply scattered light in tissues. The DAC architecture, which utilizes off-axis and separated beam paths for illumination and detection, has previously been shown to be superior to single-axis confocal (SAC) microscopy for the spatial filtering (rejection) of unwanted background light. With the MAD approach, a modulation of the alignment between the illumination and collection beam paths tags ballistic photons emanating from the focal volume with a characteristic radio frequency that can be extracted and separated from background signal using lock-in detection. We report here an optimized form of MAD confocal microscopy where we have fully mitigated tradeoffs in performance in an initial proof-of-concept system in order to recover the imaging speed of DAC microscopy while retaining contrast enhancement of 6 dB (signal-to-background ratio) with a secondary improvement in optical-sectioning and in-plane resolution. Validation is demonstrated with light-scattering tissue phantoms and freshly excised tissues.