In this report, we present implementation and validation of machine-learning classifiers for distinguishing between cell types (HeLa, A549, 3T3 cell lines) and states (live, necrosis, apoptosis) based on the analysis of optical parameters derived from cell phase images. Validation of the developed classifier shows the accuracy for distinguishing between the three cell types of about 93% and between different cell states of the same cell line of about 89%. In the field test of the developed algorithm, we demonstrate successful evaluation of the temporal dynamics of relative amounts of live, apoptotic and necrotic cells after photodynamic treatment at different doses.

Digital holographic microscopy supplemented with the developed cell segmentation and machine learning and classification algorithms is implemented for quantitative description of the dynamics of cellular necrosis induced by photodynamic treatment in vitro. It is demonstrated that the developed algorithms operating with a set of optical, morphological, and physiological parameters of cells, obtained from their phase images, can be used for automatic distinction between live and necrotic cells. The developed classifier provides high accuracy of about 95.5% and allows for calculation of survival rates in the course of cell death.

We report a general theory of the two-photon excited fluorescence (TPEF) of rotating molecules under the condition of anisotropic depolarisation. The obtained expressions for the TPEF intensity are based upon the spherical tensor approach and valid for any symmetric, or asymmetric top molecule and for any photon polarisation. The expressions are written in terms of the molecular parameters M _K (R, R′, t) which have clear tensor notation and contain all molecular information that can be extracted from the TPEF experiments. Two important extreme cases are discussed. If the molecular rotation period is much larger than the excited state lifetime, the obtained expressions describe the TPEF in non-rotating molecules under the condition of isotropic relaxation. The time-independent part of these expressions is in agreement with the earlier results of McClain [J. Chem. Phys., 58, 324 (1973)]. The relationship between McClain's molecular parameters and the parameters M _K (R, R′, 0) is presented and analysed. If the molecular rotation period is much smaller than the excited state lifetime, the expressions describe anisotropy of fluorescence from isolated ro-vibrational molecular states. As shown, under certain approximations the expression still can be written in terms of the M-parameters which are reduced due to molecular rotation by about a factor of (2K + 1). This result manifests that the fluorescence anisotropy is reduced, but not lost due to the molecular rotation.