A New Optical System for Detecting Collagen in Standard Tissue Sections – Without Special Stains or Second-Harmonic-Generation Microscopy.
Authors: Levenson R. , Hoyt C. 
Published: December 2010

Abstract
Background: There is considerable interest in understanding stroma-tumor interactions at both a molecular and structural level. Specifically, it appears that collagen deposition and remodeling are central processes in modulating breast cancer growth, local invasion and metastasis. However, conventional methods of visualizing collagen typically require the use of special stains (e.g. trichrome, picrosirius red) and, typically, the cutting of additional sections. Second-harmonic generation microscopy provides an alternative way of detecting collagen in tissue and tissue sections, but this is a complicated optical method requiring specific laser light sources. We present a new system (Nubrio™) that can detect collagen in conventional tissue sections. The specimens can be unstained, or stained with hematoxylin, eosin and/or conventional fluorescent or chromogenic labels.Materials and Methods: The optical method used involves birefringence imaging with polarization-based technology developed by CRI. Collagen molecules have repeating elements that interact anisotropically with polarized light in such a way that their abundance and orientation can be readily measured. This capability has been combined with liquid crystal tunable filters in a single enclosure so that the high-resolution collagen signals are perfectly superimposed on color or multispectral images from standard histology specimens.Results: Streamlined software allows for the capture of monochrome, color, multispectral and birefringence (retardance) images, and for their display in multilayer image stacks (where the display status of each layer can be separately controlled). Apart from the Nubrio detection system, which connects to standard microscopes via a C-mount adaptor, the only modification required is the placement of a circular polarizer in the illumination light path, typically locating it over the microscope condenser.After setting the image acquisition parameters and taking appropriate background images for correct retardance and flat-fielding purposes, subsequent multimodal, multilayer images can be acquired with a single command. It takes approximately 30 seconds to acquire a multispectral image, a retardance image, to construct a data stack and display the resulting composite image, which can be quite striking in appearance. The orientation and abundance of collagen can be assessed at low or high magnification, and the interactions of cancer cells and invasive and non-invasive epithelial cell clusters with the stroma can be readily monitored. Apart from signals arising from muscle (also a birefringent tissue element) these optical measurement correlate well with trichrome staining results and appear to generate higher spatial resolution.Discussion: The Nubrio system is well-suited for investigating tumor-stroma interactions, connective tissue invasion and re-modeling, and can be combined with molecularly specific multiplexed imaging to give a more complete picture of complex biological phenotypes.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5001.