3D Gene Expression

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Overview:
   Summary
   Project Goals

Methods:
   Staining & Mounting
   Probe Constructs
   Staging
   Imaging

Software:
   PointCloudXplore
   PointCloudToolBox
   PointCloudAlign
   NODE

File Formats:
   PointCloud (.pce)
   VirtualEmbryo (.vpc)
   Correspondence files

Imaging

The embryos are mounted and imaged whole with a Zeiss 510 META/NLO confocal laser-scanning microscope with a plan-apochromat 20x, 0.75 numerical aperture objective. This objective allows imaging of entire embryos in a single field-of-view while providing sufficient resolution and sensitivity for the subsequent analyses. The three fluorophores are excited simultaneously with a 750nm laser line from a Coherent Chameleon laser. Two-photon excitation yields better z-resolution and deeper penetration into tissue than single-photon excitation. The resulting emission spectra is split by dichroic mirrors and collected by three independent photomultiplier tubes (PMTs). The signals are digitized into 12 bits and recorded as three-channel images, each of size up to 1024 by 1024 by 150 pixels, which varies depending on the embryo size and orientation. Each pixel had a transverse dimension of 0.45m and an axial dimension of approximately 1.6m, which varies slightly with the refractive index of the mounting medium. The gain and offset of the PMTs are set for each image individually so that all the pixels of interest fall within the 12-bit dynamic range.

Before acquisition the embryos are viewed under phase contrast to determine their developmental stage.

Because of their large size (0.3-0.5 Gb each) the 3D confocal image stack files are not available from this public database, but are available by request.