Abstract
Light sheet fluorescence microscopy (LSFM) is rapidly becoming an essential technology for mesoscopic imaging of samples such as embryos and adult mouse organs. However, LSFM can suffer from optical artifacts for which there is no intrinsic solution. The attenuation of light due to absorbing material causes “shadow” artifacts along both the illumination and detection paths. Several approaches have been introduced to reduce this problem, including scanning illumination and multi-view imaging. However, neither of these approaches completely eliminates the problem. If the distribution of the absorbing material is complex, shadows cannot be avoided. We introduce a new approach that relies on multi-modal integration of two very different mesoscopic techniques. 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. Here, we demonstrate a hybrid instrument (OPTiSPIM) that can quantify this attenuation and use the information to correct the shadow artifacts of LSFM.
Highlights
In recent years, techniques for imaging threedimensional (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
In an approach similar to that used by Vinegoni et al.[43] to improve fluorescence optical projection tomography (OPT) reconstructions, we explore whether an accurate 3D map of attenuation can be used to computationally correct the shadow artifacts generated by standard Light sheet fluorescence microscopy (LSFM) imaging
In LSFM, the camera directly images optical sections illuminated by the light sheet; pixel values typically map directly into the 3D data set
Summary
Techniques for imaging threedimensional (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. 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. One of the advantages of OPT is that it can be used for fluorescent (fluorescent proteins and fluorophore-labeled antibodies) and non-fluorescent (natural pigmentations and colored dyes) contrasts
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