Morphology of Apoptotic, necrotic and oncotic cells.

<p><b>A.</b> Characteristic apoptotic, necrotic and oncotic cells in multimodal holographic microscope, simulated DIC (differential interference contrast). 20 × magnification was used in MHM. Annexin V staining for the verification of cell membrane alteration. Red arrow indicates annexin V-positive “advanced” oncotic cell. Apoptotic cells displayed in initial step (left) with the typically round-shaped cells and in advanced step with the formation of apoptotic bodies. <b>B.</b> Scheme of typical apoptotic, necrotic and oncotic cells. Typical characteristics visible by MHM phase image. For a detailed description of the characteristic features of apoptotic, necrotic, and oncotic cells, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121674#pone.0121674.t001" target="_blank">Table 1</a>.</p

Characteristic features of apoptosis, oncosis, and necrosis.

<p>Characteristic features of apoptosis, oncosis, and necrosis.</p

Estimation of oncosis progression by multimodal holographic microscopy.

<p>Annexin V (green) and propidium iodide (PI, red) staining. Initial step of oncosis (first row, red arrow) is annexin V−/PI− and thus distinguishable only by native morphology, see typical cytoplasmic bleb in the phase image. This causes false-negativity by flow-cytometry. Second, early oncotic cells feature larger blebs and are annexin V+/PI−. Late oncosis is double positive for staining, with no apparent karyolysis. Advanced oncosis/necrosis transition is typical by double-positive staining and karyolysis.</p

Holographic mode setup in Multimodal holographic microscope is based on the Mach-Zehnder-type interferometer.

<p>The light is divided into two separate optical paths—object arm and interferometer reference arm. Both arms consist of condenser (C), objective (O) and tube lens (TL). In the reference arm, a diffraction grating (DG) is placed. The object beam and the reference beam recombine in the output plane and create interference fringes pattern, which is captured by the camera (D). S—source; CL—collector lens; BS—beam splitter; M—mirror; C—condenser; O-objective; TL—tube lens; DG—diffraction grating; OL—output lens; D—detector.</p

Characteristic apoptotic, necrotic and oncotic cells in transmission electron microscope (TEM).

<p><b>A</b>. Apoptotic cell, overall view, 2800 ×. <b>B</b>. necrotic cell, 2800 ×. <b>C</b>. oncotic cell, 2800 ×, <b>D</b>. Detail of apoptotic cell nucleus, 5600 ×. <b>E</b>. Detail of necrotic cell, 14000 ×. <b>F</b>. Detail of oncotic cell cytoplasm, 11000 × magnification. Red arrow—nuclear fragmentation. Black arrow—rupture of plasmatic membrane. White arrow—karyolysis. Yellow arrow—reticular nucleolus. Dark blue arrow—dilatation of nucleus. Green arrow—dilatation of ER and Golgi. Pink arrow—cytoplasmic bleb. Light blue arrow—chromatin condensation. Violet arrow—formation of cytoplasmic vacuoles. Orange arrow—initial lysis of nucleolus. Brown arrow—mitochondrial swelling.</p

Differences in cell death estimation between flow cytometry and holographic microscopy.

<p><b>A.</b> Plumbagin treatment, no annexin V/PI staining, used for gating set-up. Upper dot plot indicates annexin V/PI fluorescence, lower dot plot indicates FSC/SSC of these cells colour-coded according to gating regions. <b>B.</b> Annexin V/PI staining, untreated cells. 92% are double negative for staining. <b>C.</b> Annexin V/PI staining after 3 h of the experiment. See increased populations of annexin V-positive (green gating) and double positive (red staining). In FSC/SSC scatter plot, arrows indicate two populations (gating regions) of annexin V+/PI− cells: (R1) smaller cells (lower FSC) and (R2) larger cells (higher FSC). See the Results section for details. <b>D.</b> Multimodal holographic microscope, phase image. 10 × magnification 3 h after 2 μM plumbagin treatment. Red-outlined cells show size increase and oncotic phenotype, green-outlined cells show surface area decrease and apoptotic phenotype, blue-outlined cells show no changes during 2 h monitoring. For typical morphological criteria of oncotic/apoptic cells see (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121674#pone.0121674.t001" target="_blank">Table 1</a>) <b>E</b>. Relative change of cell surface in individual cells (relative to initial time point). Colour coding of individual cells is based on (D). <b>F</b>. Mass of individual cells in pg. Note a significantly higher mass of the “decrease-size” cell population. <b>G</b>. Time-lapse of typical oncotic “increase size” cell indicated by red arrow in (D), simulated digital interference microscopy <b>H.</b> Time-lapse of typical “decrease size” apoptotic cell indicated by green arrow in (D). Simulated digital interference microscopy. FSC—forward scatter, SSC—side scatter, PI—propidium iodide.</p