Confocal fluorescence microscope with dual-axis architecture and biaxial postobjective scanning

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Abstract
Confocal microscopy is a powerful tool for imaging in biological tissue, and can be used in vivo if the lenses and scanning mechanisms can be made sufficiently small. This method performs optical sectioning in a highly scattering media with subcellular resolution, a high signal-to-noise ratio (SNR), and a good contrast ratio,1 an achievement not easily matched by any other imaging modality. Also, molecular structures can be identified by fluorescence markers to reveal previously unobservable details about biological processes in vivo. In addition, a microscope implanted in an animal model can perform longitudinal studies over time, thus preserving experimental continuity and reducing the number of sacrificed animals. Currently, in vivo confocal imaging is performed on exposed tissue surfaces such as that of skin or from tissue exposed surgically.2 3 4 5 6 Internal access to tissue is limited by the large physical dimensions of conventional microscopes objectives. A numerical aperture (NA) of at least 0.75 is required to achieve an axial resolution of a few micrometers, the dimensions necessary to resolve molecular and subcellular structures.7 In the conventional single-axis configuration, the same lens is used for illumination and image collection, and the optics cannot be reduced to millimeter scale without sacrificing resolution, field of view (FOV), or working distance (WD). Also, because the scanning mechanism is located proximal to the objective, multiple optical elements are required to correct for aberrations and achieve high performance. Furthermore, the use of standard objectives in vivo is rather cumbersome.