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Quantitative Immunostaining: 3D X‐ray Microscopy for Visualizing and Measuring Protein Distribution in Three Dimensions
Author(s) -
Merkle Arno,
Metscher Brian,
Yun Sylvia,
Lau SH
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.532.6
Subject(s) - microscopy , immunostaining , optical sectioning , microscope , biological specimen , optics , light sheet fluorescence microscopy , materials science , tomography , resolution (logic) , scanning confocal electron microscopy , pathology , computer science , physics , artificial intelligence , immunohistochemistry , medicine
Immunostaining has become an important tool for biological and biomedical research, providing information on the distribution of gene products such as proteins and ribonucleic acids, and has been primarily developed through optical microscopy‐based techniques, including serial sectioning histology and optical projection tomography (OPT). However, due to the limited penetration depth of visible light, these techniques are often limited to either small samples ranging from tens to hundreds of microns or to lower resolutions (10 μm). We describe a three‐dimensional (3D) imaging technique using a laboratory based 3D X‐ray microscope (XRM) for quantitative characterization of proteins in small animal embryos at high resolutions of <1 μm spatial resolution (<200 nm pixel). By using non‐destructive X‐rays that achieve large sample penetration, we demonstrate whole mount imaging capabilities of antibody probes in developing embryos. We will additionally discuss software techniques to measure X‐ray attenuation within the sample to provide quantitative mapping of protein densities. Future directions include correlative studies using compatible molecular probes for light and electron microscopy and counterstaining organs and cells with additional heavy metal stains.