Optical Projection Tomography as a Tool for 3D Microscopy and Gene Expression Studies

Abstract

Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.

Unleashing Optics and Optoacoustics for Developmental Biology

“…The technique was originally termed optical projection tomography [14][15][16], and was first published as a technique capable of imaging cleared and fixed tissues, but was later also applied to microscopic samples in vivo [17][18][19][20]. Image formation is based on using mathematical models to reconstruct optical contrast within the medium being imaged, an approach capable of imaging fluorescence and absorption.…”

Assembling models of embryo development: Image analysis and the construction of digital atlases

“…OPT (Sharpe et al, 2002) was introduced as an alternative optical method to fluorescence microscopy and overcomes the limitation of the specimen thickness. OPT generates data by acquiring many views of the same specimen at different rotation angles then assembled to create a 3D volume (Fig.…”

Multimodal imaging of mouse development: Tools for the postgenomic era

“…A second method used for 3D reconstruction of embryonic tissue is optical projection tomography (OPT; Sharpe et al, 2002; Table 1). OPT can also record 3D expression patterns and has the added advantage that the embryo is left in tact.…”

“…Improvements to reconstruction algorithms recently have been reported that decrease the contribution from various types of noise associated with image acquisition (Walls et al, 2005). Scatter still reduces the spatial resolution one can achieve with this method (Sharpe et al, 2002), compared with other forms of light microscopy, but the benefit of having a complete 3D volume from a sample 1-10 mm thick (typical; several centimeters maximum, R. Henkelman, personal communication) is worth this sacrifice in many cases.…”