OPT has been implemented for both fixed tissue 7, 8, 9 and live imaging 10, 11, 12, 13, and for applications such as developmental biology 14, diabetes studies 8, and immunology 15. OPT collects a series of projection images of the sample from different angles and computationally reconstructs a 3D image of the sample using filtered back-projection or algebraic reconstruction techniques 6. One of the first mesoscopic techniques to fill this “imaging gap” was optical projection tomography (OPT) 5-an optical implementation of computed tomography that is analogous to X-ray computed tomography. At the other end of the scale, macroscopic imaging methods, such as magnetic resonance imaging (the abbreviations and symbols used in this paper are summarized in Supplementary Table 2), are well suited for whole organisms ranging from rats to humans however, they have lower a resolution and reduced power to visualize specific molecular labels. Both traditional microscopy 1 and recently developed “nanoscopy” methods 2, 3, 4 are well suited to single cells or small groups of cells but are not optimal for imaging larger samples, such as fly, fish, and mammalian embryos, or intact organs of adult model systems such as the mouse brain, lung, or pancreas. In recent years, techniques for imaging three-dimensional (3D) mesoscopic samples-those ranging in size from tens of microns to more than a centimeter-have emerged to fill a previously unoccupied niche in the field of biological imaging. Here, we demonstrate a hybrid instrument (OPTiSPIM) that can quantify this attenuation and use the information to correct the shadow artifacts of LSFM. Unlike LSFM, optical projection tomography (OPT) can operate in transmission mode to create a voxel map of the 3D distribution of the sample’s optical attenuation. We introduce a new approach that relies on multi-modal integration of two very different mesoscopic techniques. If the distribution of the absorbing material is complex, shadows cannot be avoided. However, neither of these approaches completely eliminates the problem. Several approaches have been introduced to reduce this problem, including scanning illumination and multi-view imaging. The attenuation of light due to absorbing material causes “shadow” artifacts along both the illumination and detection paths. However, LSFM can suffer from optical artifacts for which there is no intrinsic solution. Light sheet fluorescence microscopy (LSFM) is rapidly becoming an essential technology for mesoscopic imaging of samples such as embryos and adult mouse organs.

Attenuation artifact meaning