Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution

Several well-established techniques are available to obtain 3-D image information of biomedical specimens, each with their specific advantages and limitations. Orthogonal plane fluorescence optical sectioning (OPFOS), or selective plane illumination microscopy (SPIM), are additional techniques which, after adequate specimen preparation, produce high quality, autoaligned sectional images in nearly real time, of bone as well as soft tissue. Up until now, slicing resolutions down to $14μm$ have been obtained. We present a high resolution (HR) OPFOS method, which delivers images that approach the quality of histological sections. With our HROPFOS technique, we achieve in-plane resolutions of $1μm$ and a slicing resolution of $2μm$. A region of interest within an intact and much larger object can be imaged without problems, and as the optical technique is nondestructive, the object can be measured in any slicing direction. We present quantitative measurements of resolution. A 3-D model reconstructed from our HROPFOS data is compared to SEM results, and the technique is demonstrated with section images and 3-D reconstructions of middle ear specimens.