Collagen can be visualized using a number of meth ods. Routine techniques such as histology (hematoxylin eosin staining), histochemistry (van Gieson staining, picrosirius red, etc.), immunohistochemistry, and elec tron microscopy are the most accessible and can be per formed in virtually any biological laboratory. On the other hand, they require multistep treatment of tissue samples and can cause undesirable morphological changes in the extracellular matrix, of which collagen is a predominant component. However, the most essential limitation of these techniques is that they are performed ex vivo. Some techniques, e.g., smallangle Xray scattering [1, 2] and magnetic resonance imaging (MRI) [3, 4], allow collagen visualization in real time without tissue pretreatment and can be potentially used for in vivo monitoring, although they require expensive and technically sophisticated equipment. Another method that has recently attracted a considerable interest of researchers is nonlinear microscopy based on the second harmonic generation (SHG) phenomenon. SHG microscopy is a highly specif ic optical method of direct visualization of extracellular fibrillar collagen that can be carried out using most two photon fluorescence microscopes [5, 6]. SHG microscopy is a noninvasive technique (i.e., does not involve phototoxicity and photobleaching); it does not require additional contrasting agents for collagen moni toring, and, hence, can be readily used as an auxiliary technique in in vivo studies or as an alternative to the tra ditional methods mentioned above. Despite the fact that most SHG microscopy studies of collagen have been con ducted ex vivo so far, this method offers tremendous potential for in vivo examination, including in clinical practice [79]. Examination of connective tissue based on the SHG images requires quantitative processing in order to mini mize interpretation errors and ensure unbiased compari ISSN 00062979, Biochemistry (Moscow), 2019, Vol. 84, Suppl. 1, pp. S89S107. © Pleiades Publishing, Ltd., 2019. Russian Text © V. V. Dudenkova, M. V. Shirmanova, M. M. Lukina, F. I. Feldshtein, A. Virkin, E. V. Zagainova, 2019, published in Uspekhi Biologicheskoi Khimii, 2019, Vol. 59, pp. 181218. REVIEW S89 Abbreviations: FFT, fast Fourier transform; FOS, firstorder statistics; GLCM, gray level cooccurrence matrix; ROI, region of interest; SHG, second harmonic generation; SOS, second order statistics. * To whom correspondence should be addressed. Examination of Collagen Structure and State by the Second Harmonic Generation Microscopy V. V. Dudenkova 1 , M. V. Shirmanova 1 , M. M. Lukina 1 , F. I. Feldshtein 1 , A. Virkin 2 , and E. V. Zagainova 1,a * 1 Scientific Research Institute of Biomedical Technologies, Privolzhsky Research Medical University, 603104 Nizhny Novgorod, Russia 2 Ontario Cancer Institute, University Health Network, Toronto, Canada a email: orannge@mail.ru Received August 8, 2018 Revised September 13, 2018 Accepted September 13, 2018 Abstract—Collagen is the major component of the extracellular matrix in mammals and its characteristics provide impor tant information about the state of connective tissue. There are only few methods of labelfree visualization of collagen fibers; the most frequently used is the second harmonic generation (SHG) microscopy. SHG microscopy is a noninvasive technique for the assessment of the abundance and structure of fibrillar collagen with a high resolution and specificity. At constant measurement parameters (magnification, excitation power, resolution, digital gain of registration matrix), quanti tative analysis of SHG images provides a reliable characterization of collagen state. Current approaches to the SHG signal quantification are numerous and typically should be adapted to a specific task. In this review, we systematize the variety of these approaches and present the examples of biomedical application of the SHG signal quantitative analysis, as well of combined application of SHG and autofluorescence imaging. DOI: 10.1134/S0006297919140062 Keywords: second harmonic generation, collagen, quantitative analysis, first order statistics, gray level cooccurrence matrix, fast Fourier transform