Original Article Association between Chromatin Structural Organization of Peripheral Blood Neutrophils and Self-Perceived Mental Stress: Gray-Level Co-occurrence Matrix Analysis Nikola Topalovic 1 , Sanja Mazic 1 , Dejan Nesic 1 , Olivera Vukovic 2 , Jelena Cumic 3 , Darko Laketic 4 , Ivana Stasevic Karlicic 5 and Igor Pantic 1,6 * 1 University of Belgrade, Faculty of Medicine, Institute of Medical Physiology, Visegradska 26/II, RS-11129, Belgrade, Serbia; 2 University of Belgrade, Faculty of Medicine, Institute of Mental Health, Palmoticeva 37, RS-11000, Belgrade, Serbia; 3 University of Belgrade, Faculty of Medicine, University Clinical Centre of Serbia, Dr. Koste Todorovica 8, RS-11129, Belgrade, Serbia; 4 University of Belgrade, Faculty of Medicine, Institute of Anatomy, Dr Subotica 4/2, RS-11129, Belgrade, Serbia; 5 Clinic for Mental Disorders Dr Laza Lazarevic, Visegradska 26, RS-11129, Belgrade, Serbia and 6 University of Haifa, 199 Abba Hushi Blvd. Mount Carmel, Haifa IL- 3498838, Israel Abstract Methods based on the evaluation of textural patterns in microscopy, such as the “gray-level co-occurrence matrix” (GLCM) analysis are modern and innovative computer and mathematical algorithms that can be used to quantify subtle structural changes in cells and their organelles. Potential application of GLCM method in the fields of psychophysiologyand psychiatry to this date has not been systematically investigated. The main objective of our study wasto test the existence and strength of the association between chromatin structural orga- nization of peripheral blood neutrophils and levels of self-perceived mental stress. The research was done on a sample of 100 healthy student athletes, and the Depression, Anxiety, and Stress Scales (DASS-21) were used for the estimation of psychological distress. Chromatin textural homogeneity and uniformity were negatively correlated ( p < 0.01) with mental distress and had relatively good discriminatory power in differentiating participants with normal and elevated stress levels. As an addition, we propose the creation of a machine learning model based on binomial logistic regression that uses these and other GLCM features to predict stress elevation. To the best of our knowledge, these results are one of the first to establish the link between neutrophil chromatin structural organization quantified by the GLCM method and indicators of normal psychological functioning. Key words: microscopy, morphology, neutrophil, nucleus, texture (Received 8 June 2021; revised 27 June 2021; accepted 14 July 2021) Introduction During the last two decades, there has been an accelerated devel- opment of information technologies in medicine and biology, which has led to the emergence of numerous methods for signal analysis. Many of these methods have potential applications as diagnostic aids in pathology, internal medicine, and neurology. Of particular importance are computer techniques for the analysis of images such as digital micrographs obtained by light and elec- tron microscopy. In this way, histological and pathological param- eters of cells and tissues, such as heterogeneity and uniformity, can be quantified and compared, which in the future could signif- icantly contribute to the contemporary diagnostic procedures (Biran et al., 2017; Chang & Mrkonjic, 2020; Thakur et al., 2020; Turk & Waller, 2020). Methods based on the evaluation of textural patterns, such as the “gray-level co-occurrence matrix” (GLCM) analysis, can be used to quantify structural changes in cells and their organelles in physiological and pathological conditions (Tan et al., 2016; Dhruv et al., 2019; Dincic et al., 2020). Also referred to as co-occurrence matrix or co-occurrence distribution, the GLCM applies complex computational calculations to the pairs of gray- scale values assigned to resolution units within the digital micro- graph. Some of the most important GLCM features include angular second moment (ASM) as an indirect indicator of tex- tural uniformity and inverse difference moment (IDM) as a mea- sure of textural homogeneity. In the past, this technique has been used several times to evaluate structural changes in nuclear chro- matin, during the aging process and programmed cell death (Pantic et al., 2016b; Nikolovski et al., 2019; Pantic et al., 2020; Davidovic et al., 2021). It has been shown that certain textural parameters of chromatin change as the result of the action of numerous chemical substances and biologically active com- pounds. Also in the field of neuroscience, GLCM analysis can be used to assess the consequences of trauma to the cerebral cortex, as well as certain physiological and morphological *Corresponding author: Igor Pantic, E-mail: igorpantic@gmail.com; igor.pantic@ med.bg.ac.rs Cite this article: Topalovic N, Mazic S, Nesic D, Vukovic O, Cumic J, Laketic D, Stasevic Karlicic I, Pantic I (2021) Association between Chromatin Structural Organization of Peripheral Blood Neutrophils and Self-Perceived Mental Stress: Gray- Level Co-occurrence Matrix Analysis. Microsc Microanal. doi:10.1017/ S143192762101240X © The Author(s), 2021. Published by Cambridge University Press on behalf of the Microscopy Society of America Microscopy and Microanalysis (2021), 1–7 doi:10.1017/S143192762101240X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65