Development of high-speed, integrated high-resolution optical coherence microscopy and dual-channel fluorescence microscopy for the simultaneous co-registration of reflectance and fluorescence signals

Optical coherence microscopy is an imaging technique capable of generating three-dimensional images of structures in tissue, but with limited functional imaging ability. Fluorescence microscopy offers functional information of cellular activities. The integration of both imaging modalities can enable the evaluation of the impact of structural information on cells activities. To develop an integrated system combining high-resolution optical coherence microscopy (HR-OCM) and dual-channel scanning confocal fluorescence microscopy (DC-SCFM) to enable the coregistration of structural and functional information. Different strategies to enable the simultaneous recording of information, as well as to overcome the focal plane mismatch between both imaging modalities were developed. The system's performances were evaluated in imaging fluorescence microspheres embedded in multi-layer tape and silicone phantom. HR-OCM achieved an axial resolution of 2.4-mu m in tissue over an imaging depth of 1-mm. A speed of 250 kHz and a lateral resolution of 2-mu m over a field-of-view of 1.1 mm x1.1 mm were demonstrated with the combined system. DC-SCFM allowed to discriminate between green and red microspheres while HR-OCM enabled their depth localization. The combined system is synergistic in generating structural and functional information of samples, and has the potential to enable new investigations of biological processes in animal models.