Performance of dual axes confocal microscope for in vivo molecular and cellular imaging

The application of conventional confocal microscopes with high numerical aperture (NA) to in vivo imaging is limited by the objectiveis large physical dimensions and short working distance. We are developing a confocal microscope that uses simple low NA lenses oriented in a dual axes configuration for miniaturization and in vivo imaging. This architecture achieves a long working distance, micron level axial resolution, and reduced noise from scattered light outside the focal volume. Combined with the novel method of post-objective scanning, this design can be scaled down to millimeter dimensions. We derive the dual axes response from diffraction theory, and construct two tabletop prototypes to demonstrate the performance of this approach. We collect images from freshly excised biopsy specimens of human esophagus and transgenic mouse cerebellum expressing GFP. With horizontal cross-sectional images, we achieve 1 to 2 μm resolution and collect reflectance and fluorescence images. With vertical cross-sectional images, we achieve 4 to 5 μm resolution, dynamic range of 70 dB, and tissue penetration over 1 mm. An instrument miniaturized with this configuration could be used for in vivo cellular and molecular imaging.