Method of detecting fibrous tissue in a biological specimen using co-localized images generated by second harmonic generation and two photon emission

An image generating and analyzing system for quantifying presence of collagen fibers in a biological test specimen comprising:

• a platform for deposition of the test specimen thereon;
• an electromagnetic light source coupling to the platform to direct a radiation at an excitation wavelength to an interested region of the test specimen, the radiation resulting in emission of a first and a second electromagnetic signals respectively caused by two-photon excitation (TPE) of excitable material and second harmonic generation (SHG) of optically non-linear materials in the collagen fibers in the test specimen;
• a first sensor and a second sensor being arranged to respectively real-time record the emitted first and second electromagnetic signals and convert the received first and second electromagnetic signals separately into a first digital signal and a second digital signal;
• an optical assembly for manipulating the radiation directed to the test specimen and the emitted first and second electromagnetic signals prior to having the first and second electromagnetic signals recorded by the first and second sensors;
• a microprocessor unit being configured to use the first and second electromagnetic signals in generating a co-localized image having a set of image properties and being indicative of spatial distribution of the collagen fibers in the interested region, quantify one or more scalar features and distribution features of the co-localized image to generate quantified result for each scalar and/or distribution feature, quantifying collagen deposition in the test specimen using the quantified results for the extracted scalar and distribution features;
• and
• a visual display, in communication with the microprocessor unit, providing an interface comprising one or more modules for customizing the set of adjustable parameters, displaying the co-localized image, quantified results of the scalar features and distribution features, and quantified collagen deposition.