A Study on Image Quality in Polarization-Resolved Second Harmonic Generation Microscopy

Abstract

Second harmonic generation (SHG) microscopy represents a very powerful tool for tissue characterization. Polarization-resolved SHG (PSHG) microscopy extends the potential of SHG, by exploiting the dependence of SHG signals on the polarization state of the excitation beam. Among others, this dependence translates to the fact that SHG images collected under different polarization configurations exhibit distinct characteristics in terms of content and appearance. These characteristics hold deep implications over image quality, as perceived by human observers or by image analysis methods custom designed to automatically extract a quality factor from digital images. Our work addresses this subject, by investigating how basic image properties and the outputs of no-reference image quality assessment methods correlate to human expert opinion in the case of PSHG micrographs. Our evaluation framework is based on SHG imaging of collagen-based ocular tissues under different linear and elliptical polarization states of the incident light.Second Harmonic Generation (SHG) microscopy is regarded nowadays as a very useful and powerful tool for characterizing biological tissues 1,2 . Its potential originates from the ability to image in a label-free manner non-centrosymmetric structures, which exhibit a non-vanishing second-order susceptibility tensor χ (2) . Under the influence of an external electric field, such structures generate a nonlinear optical signal at exactly half the wavelength of the excitation source. This emission can be easily isolated from the excitation wavelength or from associated fluorescence signals by using spectral filters. Most SHG applications that focus on physiological assessment and disease diagnostics rely on imaging type-I collagen 3-6 , the most abundant protein in the human body and the main structural protein in the extracellular matrix of animal tissues. The investigation of collagen distribution in tissues with SHG enables a precise and non-invasive assessment of extracellular matrix modifications, which represent a hallmark for a wide range of pathologies, including cancer 7,8 . Polarization-resolved SHG (PSHG) microscopy extends the potential of SHG, by exploiting the fact that this nonlinear signal is sensitive to the polarization state of the excitation beam 9 . In the case of collagen-based tissues, the SHG emission depends on the alignment between the collagen fibers/fibrils and the polarization of the excitation light [10][11][12] . The additional dimension available in PSHG data sets can be used to analyze the optical anisotropy and hence to better probe the molecular organization and the external arrangement of collagen-based structures [10][11][12][13][14][15][16] . With the hierarchical organization of collagen being closely intertwined with the biophysical, mechanical and hence functional properties of most tissues, PSHG's potential can be steered towards finding answers to important questions over diseases genesis, progression and treatment. Moreover, PSHG can potentially be used ...

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