The application of conventional confocal microscopes with high numerical aperture (NA) to in vivo imaging is limited
by the objectiveís 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.
We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution 4.4 µm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging.