We have studied the interaction of the DNA molecule with the ligand 3,3'-diaminobenzidine (DAB) by performing single molecule stretching experiments with optical tweezers and dynamic light scattering (DLS) on the DNA-DAB complexes. In the stretching experiments, the persistence and contour lengths of the complexes were measured as a function of DAB concentration, allowing one to infer the main binding mechanism and also to determine the physicochemical parameters of the interaction. In the DLS experiments, the effective size of the complexes, measured as the hydrodynamic radius, was monitored as a function of DAB concentration. We found a qualitative agreement between the results obtained from the two techniques by comparing the behaviors of the hydrodynamics radius and the radius of gyration, since this last one can be expressed as a function of the persistence and contour lengths.

Precise diagnosis and prognosis are key in prevention and reduction of morbidity and mortality in all types of cancers. Here we show that changes in the collagen fibres in the main histological subtypes of canine mammary gland carcinomas are directly associated with the tumour behaviour and the animal survival time and could become a useful tool in helping with diagnosis. Imaging by second harmonic generation and multiphoton excited fluorescence microscopy were performed to evaluate the collagen and cellular segment parameters in cancer biopsies. We present a retrospective study of 45 cases of canine mammary cancer analysing 836 biopsies regions including normal mammary gland tissue, benign mixed tumours, carcinoma in mixed tumour, carcinosarcoma, micropapillary carcinoma and solid carcinoma. The image analyses and the comparison between the tumour types allowed to assess the collagen fibre changes during tumour progression. We demonstrate that the collagen parameters correlate with the clinical and pathological data, the results show that in neoplastic tissues, the collagen fibres are more aligned and shorter as compared to the normal tissues. There is a clear association of the mean fibre length with the dogs survival times, the carcinomas presenting shorter collagen fibres indicate a worse survival rate.

We present nonlinear microscopy imaging results and analysis from canine mammary cancer biopsies. Second harmonic generation imaging allows information of the collagen structure in the extracellular matrix that together with the fluorescence of the cell regions of the biopsies form a base for comprehensive image analysis. We demonstrate an automated image analysis method to classify the histological type of canine mammary cancer using a range of parameters extracted from the images. The software developed for image processing and analysis allows for the extraction of the collagen fibre network and the cell regions of the images. Thus, the tissue properties are obtained after the segmentation of the image and the metrics are measured specifically for the collagen and the cell regions. A linear discriminant analysis including all the extracted metrics allowed to clearly separate between the healthy and cancerous tissue with a 91%-accuracy. Also, a 61%-accuracy was achieved for a comparison of healthy and three histological cancer subtypes studied.

In this work, we use force spectroscopy to investigate the interaction between the DAPI fluorescent dye and the λ-DNA molecule under high (174 mM) and low (34 mM) ionic strengths. Firstly, we have measured the changes on the mechanical properties (persistence and contour lengths) of the DNA-DAPI complexes as a function of the dye concentration in the sample. Then, we use recently developed models in order to connect the behavior of both mechanical properties to the physical chemistry of the interaction. Such analysis has allowed us to identify and to decouple two main binding modes, determining the relevant physicochemical (binding) parameters for each of these modes: minor groove binding, which saturates at very low DAPI concentrations ( CT ∼ 0.50 μM) and presents equilibrium binding constants of the order of ∼10 M for the two ionic strengths studied; and intercalation, which starts to play a significant role only after the saturation of the first mode, presenting much smaller equilibrium binding constants (∼10 M ).

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