Bioimaging Embryology

Bioimaging & Embryology

The Bioimaging & Embryology unit focuses on functional morphology analysis using state-of-the-art 3D imaging technologies of adult mice and rats as well as tiny samples such as murine embryos and tissue samples. The anatomical annotation of skeleton dysmorphologies and developmental disorders is a key feature for interpretation of morphology phenotypes. The unit also provides the knowledge base for conditional gene inactivation during development. More info

The most prominent focus of the unit is to provide functional morphological analysis of phenotypes in adult mice and rat models and during their embryonic development.  The microCT technology provides the best cost effective approach for 3D visualization of phenotypes and the unit provides full data analysis platform 3D data processing. Besides 3D imaging, the Unit is equipped with a whole body imaging system that is suitable for imaging of fluorescence and bioluminescence reporters in mice and rats in vivo and is very advantageous especially for imaging of cancer models derived from cancer cell lines or PDX. For non-invasive imaging and cell labelling the set of lentiviral reporters is available. Beside cancer cells, the physiological processes like inflammation, kidney function or specific enzyme activity can be also non-invasively imaged.  Beside stated imaging modalities, the unit also provides experience in functional assays on primary cells or their isolation for multiOMICs technologies. The embryological tissues can be dissected and primary cell lines or organ cultures can be established as well as immortalized cell lines from knock out phenotypes can be delivered. These approaches can help to accelerate the research of mutants with embryonic lethal phenotypes.

Comprehensive morphological and functional characterization of animal models by whole-body imaging systems in vivo and ex vivo.

Standard Services MicroCT

Our microCT in vivo scanner provides sensitive and high resolution 3D imaging based on X-ray projections with voxel size 9 – 35um. Our imaging set up is suitable either for in vivo or ex vivo imaging. In vivo scans can provide comprehensive 3D visualization of bones and other mineralized tissues like teeth, but also quantification of total body fat mass and lean mass (alternative to DEXA analysis). MicroCT also provides fast imaging mode for in vivo visualization of cardiovascular system and kidneys after application of contrast agents.

Ex vivo scanning mode can be used for higher resolution imaging or use of contrast agents for imaging soft tissues (liver, kidney, hearth, neuronal tissue) or imaging fixed embryos (E9.5 – E18.5) with use of appropriate contrast agents (Iodine, PTA). For complete microCT analysis software post-processing of projection data is necessary, especially 3D reconstruction and further segmentation for visualization of morphological phenotypes. Data can be presented as static images and/or as animated 3D reconstruction movies.

Software tool box: ITK-snap, 3D-slicer, CTvox, CTanalyser, Imaris

Whole skeleton microCT scan is routinely used for unbiased morphological analysis of mouse mutant lines
Body composition analysis from 3D microCT data. Mineralized tissues like bones are in green, lean tissues like organs, intestine and muscles are in yellow and all fat tissue is in purple. Volumetric parameters can be quantified and statistically evaluated.
3D image of fixed embryo after Iodine contrasting protocol
Imaging of contrasted soft tissues is also possible with microCT. Liver were fixed and contrasted with Iodine, bile duct structure can be visualized based on differential Iodine sequestration in liver parenchyma and bile ducts.
Segmentation of teeth within the mouse skull. Such an approach can provide important information about tissues/structure morphology within the context of the whole skull or body.
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Standard Services Radiography (x-ray)

2D radiography is the best for fast analysis of mineralized/hard tissues, with combination of rotation stage can be also combined in 3D projection. This imaging modularity is mostly recommended as background image for other modularities (luminescence/fluorescence). Combination of optical imaging and X-ray is very suitable for anatomical annotation of fluorescence or luminescence signals.

X-ray imaging is used for anatomical annotation of tumour position discovered by fluorescence imaging.
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Standard services Whole Body Fluorescence Imaging

Our device is very suitable for imaging of reflected fluorescence signals which can be used for in vivo or ex vivo imaging. Even though the ex-vivo imaging provides better signal resolution, we focus mostly on application of in vivo approach in most cases. In vivo fluorescence imaging provides unique opportunities for visualization and quantification of pathological processes like inflammation, kidney function or tumour progression in longitudinal analysis.

The ability of cells to form tumours was visualized by detection of red fluorescent protein (RFP) expressed in inoculated cell line.
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Standard Services In vivo Bioluminiscence Detection

Our device includes highly sensitive camera for detection of emitted photons based on enzymatic activity like luciferase or peroxidase. This approach is suitable for in vivo imaging with emphasis on high sensitivity. This imaging is frequently used for inflammation, tumour progression, metastasis or cell homing experiments. We have successfully published our imaging protocol in the study of genetic regulation of DSS colitis. We offer our knowledge for screening of potential role of given genes in the gut regeneration processes by non-invasive imaging setup for longitudinal monitoring of healing process.

Whole body detection of luciferase reporter. Luciferase provides very high sensitivity with out non specific background, which make is suitable for less intense processes or detection of small cell populations.
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Standard Services Optical Projection Tomography (OPT)

Optical projection tomography is based on screening samples from multiple angles in fluorescent or transmitted light and subsequent 3D reconstruction from projection data. This imaging approach is very suitable for early stage embryos, tissues and organs. Samples need to be cleared first by special clearing protocols. For this purpose, we are preparing protocols for 3D imaging of LacZ stained samples, whole-mount in situ hybridization samples, fluorescent proteins presence or whole mount immunologically stained samples. For basic morphology analysis we provide rapid clearing BABB based protocol and detection of endogenous autofluorescence. Final data can be presented as static images and/or as animated 3D reconstruction movies. Software tool box: ITK-snap, 3D-slicer, Imaris.

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Standard Services Tissue Clearing and Large Z-stack Optical Sectioning

We can offer our experience with tissue clearing and whole-mount imaging protocols employed predominantly for cre dependent fluorescence reporters (R26mT/mG) since a standard data can be presented as volume rendered static images or animated 3D reconstructions.

The tooth germ from a E14.5 embryo, suitable reporter has been used, tissue clearing protocol followed by large Z-stack optical sectioning with confocal microscope. Dataset was 3D reconstructed and 3D rendered in Imaris.
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Standard services Histological Tomography

In collaboration with histology unit we can offer unique 3D visualization from serial histological sections. In this procedure, the entire organ or embryo is sectioned on histological slides, processed for hematoxylin or any other histological staining, every single section is digitalized and then 3D reconstructed in Voloom software. Datasets can be also processed in Bitplane Imaris for state of the art quantification analysis.

Whole adult kidney reconstructed from 500 serial histological sections, reconstructed in Voloom and segmented in Imaris software package.
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Standard services LacZ Staining

LacZ staining method of mouse adult tissues is important part of phenotyping screen established by IMPC. The most of knockout mouse lines generated via KOMP/ EUCOMM embryonic stem cell resources have introduced the lacZ reporter gene driven by the endogenous regulatory elements of the targeted gene. The principle of this method is histochemical staining for LacZ enzyme activity which can be detected in organs, substructures, and cells in which the gene is normally expressed. LacZ expression analysis is thus crucial for understanding of the physiological functions of a gene as well as for its role in the development or progression of diseases.

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Standard Services Embryology Services

Whole Embryo and Placenta Isolation/Dissection

Embryos can be dissected from E5.5 in following half day intervals up to E18.5. The genotyping of embryos and resorptions are among standard procedures and harvested material can delivered up to request (fresh, frozen or fixed). The embryos can be obtained also from conditionally inactivated gene models, where gene ablation design is discussed first.

Staining for ß-galactosidase Activity

Transgenic embryos and placentas carrying a lacZ expression construct can be used for whole-mount ß-galactosidase staining. From E12.5 is the staining done one dissected organs or frozen sections in order to minimize problems with substrate penetration in larger embryos.

Immunohistochemistry and In situ Hybridization Staining

Expression of RNAs or proteins can be detected using in-situ hybridization or immunohistochemistry respectively. Both can be performed either in whole mount or on sections and followed by 3D imaging methods.

Histochemical Visualization of Embryonic Skeletons

From 12.5 dpc onwards, the mouse skeleton can be visualized using the histochemical stains alcian blue (which stains the cartilaginous skeleton) and alizarin red (which stains mineralized tissues).

Embryo Imaging and Image Analysis

We use multiple imaging modalities for detail phenotype analysis in embryos of all developmental stages. We use combination of standard and high resolution microCT techniques for detail morphology and anatomy analysis, further image segmentation approaches can be used for quantification and presentation of phenotype in direct publication quality. Another imaging modality is detection of fluorescence where we use multiple tissue clearing protocols (Scal/a2, Cubic, BABB, Clarity) with combination with deep tissue imaging techniques (Light Sheet microscopy, Two-photon confocal imaging, Optical tomography and Structued illumination microscopy). For image analysis we can offer Imaris analytical station with appropriate modules for developmental biology (filament and particle tracking, Matlab extension).

Embryonic fibroblasts

Embryonic fibroblasts (MEFs) can be readily isolated from mouse embryos 9.5 dpc and older.

Cre zoo in CCP (image courtesy of Embryology unit staff)
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Custom services HREM

Survey potential interest

We are surveying interest of the scientific community in application of HREM (high resolution episcopic microscopy) in research projects. HREM is a novel 3D imaging method based on episcopic screening of serially sectioned specimens in fully automatized manner (embryos, organs). In principle, the device generates thousands of digitalized serial sections which are subsequently 3D reconstructed. The data can be presented as volume rendered static images or animated 3D reconstructions.

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