394 ISSN 1990-7931, Russian Journal of Physical Chemistry B, 2019, Vol. 13, No. 3, pp. 394–400. © Pleiades Publishing, Ltd., 2019. Russian Text © The Author(s), 2019, published in Khimicheskaya Fizika, 2019, Vol. 38, No. 6, pp. 8–14. Morphological Changes in Malignant Tumor Cells at Photodynamic Treatment Assessed by Digital Holographic Microscopy A. A. Zhikhoreva a , A. V. Belashov a, *, D. A. Gorbenko a , N. A. Avdonkina b , I. A. Baldueva b , A. B. Danilova b , M. L. Gelfond b , T. L. Nekhaeva b , I. V. Semenova a , and O. S. Vasyutinskii a a Ioffe Institute, Russian Academy of Sciences, St. Petersburg, Russia b N.N. Petrov National Medical Research Center, Ministry of Health of Russia, St. Petersburg, Russia *e-mail: belashov.andrey.93@gmail.com Received November 19, 2018; revised December 6, 2018; accepted January 21, 2019 Abstract—The paper presents an investigation of cell death dynamics and changes of cellular morphology induced by the intracellular generation of singlet oxygen as a result of photodynamic treatment. The response of kidney carcinoma, osteosarcoma, and skin melanoma cells to photodynamic treatment was analyzed through the changes in their thickness, average phase shift, and refractive index distribution. It was demon- strated that an irradiation dose of 42 J results in the necrosis of sarcoma and kidney carcinoma cells, although melanoma cells show no statistically significant changes in optical or morphological parameters. Keywords: digital holographic microscopy, singlet oxygen, morphology of living cells, necrosis, photody- namic treatment DOI: 10.1134/S1990793119030242 INTRODUCTION The increasing incidence rate of malignant diseases and the high frequency of recurrencies are the most important problems in modern oncology, requiring improvements of methods aimed at treatment of patients with this pathology. Photodynamic therapy (PDT) is one of the promising tools being used world- wide for treatment of various cancers. It uses the phys- icochemical properties of photosensitizers (PSs), which selectively accumulate in pathological tissues with increased metabolism [1] and can be activated by local irradiation with light of a wavelength corre- sponding to the absorption band of PS molecules. This activation causes generation of singlet oxygen (the first excited state a 1 Δ g of the O 2 molecule) and other reactive oxygen species that are detrimental to the tumor cells and cause tumor resorption and ablas- tics in the tumor bed [2, 3]. At present, PDT is being actively implemented in clinical practice, both as a main method and in combination with other methods for treatment of malignant neoplasms of various local- izations. In particular, it has been shown in a number of works [4–6] that this approach provides efficient treatment of cervical, esophageal, tracheal, and other cancers. A large number of photosensitizers have been developed by now [7–10], and detailed studies of their physical and chemical properties have been carried out, the most important of which are the quantum yield of reactive oxygen species, absorption spectrum, stability under irradiation, dark phototoxicity, rate of elimination from the body, and intracellular localiza- tion [11–13]. An analysis of the PS efficiency for PDT usually includes four stages: (1) investigation of photo- physical properties of the PS in solutions; (2) study of the response of malignant cells to photodynamic treat- ment with this PS in vitro and (3) in vivo; and (4) clinical study of its effectiveness in PDT. The modern approach to studies of cellular response to photodynamic treatment suggests that it is important not only to determine treatment doses and irradiation modes at which cell death occurs but also to reliably identify cell death mechanisms. Necrosis is characterized by the destruction of organelles and plasma membrane, which leads to the release of intra- cellular contents into the extracellular space and the development of inflammatory processes. Apoptosis is characterized by cell rounding, tighter packaging of the cytoplasm and organelles, followed by the intense formation of small bubbles on the plasma membrane (blebbing) and creation of individual apoptotic bodies, which are phagocytosed in vivo by macrophages or neighboring normal cells [14, 15]. The distinguishing between cell apoptosis and necrosis is usually conducted by evaluating the integ- rity of the cell membrane using standard fluorescent dyes and by analysis in a confocal fluorescent micro- scope. Other approaches have been developed, based