Long-Depth Imaging of Specific Gene Expressions in Whole-Mount Mouse Embryos with Single-Photon Excitation Confocal Fluorescence Microscopy and FISH

Abstract

Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of λF = 0.525 μm and λF = 0.580 μm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 μm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5×). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 μm has not yet been realized with two-photon fluorescence excitation.