Plum pudding random medium model of biological tissue toward remote microscopy from spectroscopic light scattering

Biological tissue has a complex structure and exhibits rich spectroscopic behavior. There is \emph{no} tissue model up to now able to account for the observed spectroscopy of tissue light scattering and its anisotropy. Here we present, \emph{for the first time}, a plum pudding random medium (PPRM) model for biological tissue which succinctly describes tissue as a superposition of distinctive scattering structures (plum) embedded inside a fractal continuous medium of background refractive index fluctuation (pudding). PPRM faithfully reproduces the wavelength dependence of tissue light scattering and attributes the "anomalous" trend in the anisotropy to the plum and the powerlaw dependence of the reduced scattering coefficient to the fractal scattering pudding. Most importantly, PPRM opens up a novel venue of quantifying the tissue architecture and microscopic structures on average from macroscopic probing of the bulk with scattered light alone without tissue excision. We demonstrate this potential by visualizing the fine microscopic structural alterations in breast tissue (adipose, glandular, fibrocystic, fibroadenoma, and ductal carcinoma) deduced from noncontact spectroscopic measurement

List of peptides observed for Vangl2 identification.

<p>List of peptides observed for Vangl2 identification.</p

List of peptides observed for Vangl1 identification.

<p>List of peptides observed for Vangl1 identification.</p

Interaction of epitope-tagged Vangl2 and Vangl1 proteins.

<p>(<b>A</b>) Schematic diagram of Vangl1 and full-length and truncated mutants of Vangl2 used for this study. (<b>B</b>) Transient co-expression of myc-Vangl1 with GFP-Vangl2, GFP-Vangl2ΔN, GFP-Vangl2ΔC, GFP-Vangl2ΔNΔC a control protein (GFP in T47D cells and immunoprecipitation with myc antibody shows co-immunoprecitation of Vangl2 with Vangl1). (<b>C</b>) Colocalization of ectopically expressed Myc-Vangl1 and GFP-Vangl2 in T47D cells and analysis by immunofluorescence and confocal analysis. All images were taken under a 40×objective. Scale bar corresponds to 10 µm and is labelled in white. (<b>D</b>) Vangl2 homodimerization is displayed by immunoprecipitation with Myc antibody shows co-immunoprecitation of Myc-Vangl2 with GFP-Vangl2. Protein complexes were separated using SDS-PAGE and western blot analysis.</p

Generation of a highly specific anti-Vangl2 monoclonal antibody.

<p>(<b>A</b>) Expression of GST, GST-NVangl1 and GST-NVangl2 fusion proteins verified by SDS-PAGE and Coomassie staining. (<b>B</b>) Selected hybridomas were screened using an ELISA against GST, GST-NVangl1 and GST-NVangl2. mAb: monoclonal antibody. (<b>C</b>) Specificity of 2G4 mAb for GST-NVangl2 shown by western blot experiments, using antibodies specific for GST, Vangl2 and Vangl1/2. (<b>D</b>) SPR analysis using 2G4 mAb (10 µg/ml) injected over immobilized GST, GST-NVangl1 or GST-NVangl2. Sensorgrams show total signal from GST fusion proteins normalised with the non-specific signal (GST).</p

Identification of an endogenous protein complex containing the Vangl2 and Vangl1 proteins.

<p>(<b>A</b>) Specificity of 2G4 mAb was verified in western blot using T47D cell extracts expressing GFP, GFP-Vangl1, Vangl2 or GFP. Proteins were separated on SDS-PAGE and analyzed with western blot with specific antibodies. Specificity of 2G4 mAb in immunoprecipitation experiments GFP-Vangl1, GFP-Vangl2 or GFP expressed in T47D cells. Western blotting was carried out using 2G4 mAb, anti-GFP and Vangl1/2 antibodies. (<b>B</b>) SKBR7 cells were treated with shLuc or shVangl2 and assessed for Vangl expression. Vangl2 was immunoprecipitated from cell lysates and detected with the same 2G4 antibody. (<b>C</b>) Proteins were subsequently separated using Bis-Tris gradient gels and silver stained. Bands specific to 2G4 mAb were excised followed by in-gel trypsin digestion, chromatographic separation and orbitrap analysis. An asterisk indicates the bands corresponding to Vangl2. (<b>D</b>) Endogenous Vangl2 and Vangl1 were identified with the presence of multiple peptides and associated with a high probability Mascot scores (at least 40 arbitrary units). Bold characters indicate the peptides identified by mass spectrometry as shown with alignment of the two sequences. Regions shaded in grey correspond to the amino-terminal regions of Vangl1 and Vangl2.</p

Differential expression of Vangl2 in different murine tissues and mouse cochlea.

<div><p>(<b>A</b>) Expression profile of Vangl2 from murine tissues (brain, kidney and lung) detected with 2G4 antibody in western blot. (<b>B</b>) Vangl2 protein expression in mouse cochlear tissue deriving from homozygous <i>Lp</i> and heterozygous Vangl2^Lp/+ heterozygous mice compared to wild-type littermates, using 2G4 mAb antibody. Corresponding densitometry measurements representing relative Vangl2 protein expression normalized to GADPH protein levels. (<b>C</b>) Absence of staining with 2G4 mAb in Vangl2 mutant. Surface views of cochleae from WT (A-A’) and Looptail homozygote (B-B’) from E17.5 mice processed for immunocytochemistry with 2G4 mAb. At this stage, the cochlea comprises a single row of inner hair cells (#) and three rows of outer Hair Cells (OHC1 *, OHC2 **, OHC3 ***) surrounded by supporting cells. Phalloidin staining (actin, red) reveals hair cells borders. The image is taken at the level of the zonula adherens for the second and third row of OHC2 and OHC3, where Vangl2 accumulates. In the WT sample, we observe accumulation of Vangl2 at the junction between hair cells and supporting cells (A-A’, arrows). In contrast, in Lp/Lp cochlea, there is a complete absence of Vangl2 staining at the membrane of the cells (B,B’). Scale bar = 3 µm. (<b>D</b>) Co-localization of staining with 2G4 mAb and Vangl1 Ab.</p> <p>Surface view of a cochlea from a newborn mouse processed for immunocytochemistry with 2G4 mAb and Vangl1 Ab. The two antibodies reveal a co-localization of Vangl1 (A”) and Vangl2 (A’”) proteins at the junction between hair cells and supporting cells (arrows). Phalloidin is in blue, Vangl1 in red, and Vangl2 in green. Scale bar = 3 µm. Note: in the green channel, remains of the tectorial membrane covering normally the cochlear epithelium lead spots of non-specific green labeling. (<b>E</b>) Schematic diagram representing the two possible models of Vangl1/Vangl2 interaction.</p></div