Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis

RESEARCH ARTICLE Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis Jan Balvan 1,2 , Aneta Krizova 2,3 , Jaromir Gumulec 1,2 , Martina Raudenska 1,2 , Zbysek Sladek 4 , Miroslava Sedlackova 5 , Petr Babula 6 , Marketa Sztalmachova 1,2 , Rene Kizek 2,7 , Radim Chmelik 2,8 , Michal Masarik 1,2 * 1Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic, 3TESCAN Brno, s.r.o., Brno, Czech Republic, 4Department of Morphology, Physiology, and Animal Genetics, Mendel University in Brno, Brno, Czech Republic, 5Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 6Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 7Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic, 8Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, Brno, Czech Republic *[email protected]ni.cz Abstract Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - “Multimodal holographic microscopy (MHM)”. The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI−phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis. Introduction Cell necrobiology is a rapidly developing field of cell biology that defines various modes of cell death pursuant to biochemical, morphological, and molecular changes accompanying distinct types of cell death including the tissue response [1]. Identification of the exact type of cell death following the cell injury is important for diagnostics, dose-response, and toxicological studies. It is extremely important to assess and interpret correctly the cellular response to severe injury including changes that occur before and after the cell death, because cell death changes could be the earliest signal of toxic reactions to a variety of drugs including the anticancer treatment. Cells can die through a number of different mechanisms inter alia by apoptosis, autophagy, necrosis, or oncosis. Nevertheless, two major PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 1/16 a11111 OPEN ACCESS Citation: Balvan J, Krizova A, Gumulec J, Raudenska M, Sladek Z, Sedlackova M, et al. (2015) Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis. PLoS ONE 10(3): e0121674. doi:10.1371/journal.pone.0121674 Academic Editor: Robert M. Hoffman, AntiCancer Inc., UNITED STATES Received: October 3, 2014 Accepted: February 3, 2015 Published: March 24, 2015 Copyright: © 2015 Balvan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: The financial support from CEITEC CZ.1.05/1.1.00/02.0068 and project for conceptual development of research organization (Faculty of Medicine, Masaryk university - ROZV/24/LF5/2014) is greatly acknowledged. Part of the work was carried out with the support of core facilities of CEITEC – Central European Institute of Technology under CEITEC-open access project, ID number LM2011020, funded by the Ministry of Education, Youth and Sports of the Czech Republic under the activity “Projects of major infrastructures for research, types of cell death are accidental cell death and programmed cell death. When assessing the major effect of a particular therapeutic drug, it is essential to know which type of cell death is involved most in the drug response. If the main mechanism involved in the cell death is oncosis followed by necrosis, the cells lose membrane integrity and release their intracellular contents, which are often aggressive, proinflammatory, and cause damage to the surrounding tissue [2]. By contrast, apoptotic cells may not promote inflammation because they are usually ingested by phagocytes before releasing their intracellular contents [3]. An important biochemical event leading to oncosis /necrosis, as opposed to apoptosis, is a rapid decrease of intracellular ATP [4,5]. The assessment of oncosis is frequently neglected, although it is an important pre-lethal phase that follows a serious cell injury and, unlike in necrosis, some mechanisms possibly exist for reversing the process [5]. Many changes typical for these two main types of cell death (accidental and programmed cell death) are detectable by flow-cytometry. Nevertheless, relying solely on the flow-cytometry could lead to the misclassification of the cell death type since—similarly as apoptotic cells— oncotic cells could exhibit external phosphatidylserine residues (PS) while maintaining membrane integrity. As a result, oncotic cells could display the annexin V+/PI−phenotype, formerly supposed to be specific of apoptotic cells [6,7]. Similarly, the TUNEL assay is also known to be non-specific for apoptosis/oncosis differentiation [8–10]. Consequently, morphological criteria are considered the most reliable evidence of apoptosis [11,12]. Characteristics of apoptosis, oncosis, and necrosis are summarized in Table 1. In this paper, we present a methodology that can be used for the rapid assessment of cell viability and distinction of oncosis and apoptosis utilizing multimodal holographic microscope (MHM). MHM combines holographic microscopy with the well-known fluorescence microscopy. The employed holographic microscopy (HM) is based on an off-axis setup with an incoherent source. In contrast with the HM laser source, the incoherent HM source enables highquality quantitative phase imaging free of speckles and parasitic interferences, comparable with the lateral resolution of conventional wide-field microscopes. Owing to the off-axis setup, only Table 1. Characteristic features of apoptosis, oncosis, and necrosis. Feature Oncosis Necrosis Apoptosis Cell size increased (swelling)*increased (swelling)*reduced (shrinkage)* Plasma membrane intact*in the early phase; increased throughput depending on the phase of oncosis disrupted*intact*; altered orientation of lipids Nucleus Nucleus dilatation*and clumping of chromatine* reticular nucleolus karyolysis*and caspase independent DNA fragmentation,lysis of nucleolus nuclear chromatin condensation*; fragmentation of DNA into nucleosome size fragments, irregularity of nucleus* Specific features swelling of organelles; membrane blebs* increasingly translucent cytoplasm*; swelling of ER and loss of ribosomes; swollen mitochondria with amorphous densities; lysosome rupture; plasma membrane rupture*; myelin figures apoptotic bodies*; pseudopod retraction*; spherical shape of cells* Energy balance ATP depletion ATP depletion retained ATP production PI/annexin V assay annexin V+/ PI−*; annexin V+/PI+* annexin V+/PI+*annexin V+/ PI−* Adjacent inflammation frequent frequent rare *indicates typical features of distinct cell death, easy observable by using MHM. ER—endoplasmatic reticulum; ATP—adenosine triphosphate; PI— propidium iodide. According to our results and [4,5,43]. doi:10.1371/journal.pone.0121674.t001 Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 2/16 development and innovations”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The cooperation with commercial company (TESCAN) does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. one hologram is needed for image reconstruction and very fast processes can be observed [13]. General characteristics of different holographic methods are summarized in Table 2. Holographic microscopy is a method of quantitative phase imaging. Providing intrinsic high contrast, phase images allow an easy segmentation of cells from the image background and monitor morphological and position changes over the time [14]. Fluorescence imaging is combined with holographic microscopy in a way that the focus plane in both methods is at the same position. This allows an easy transition between the two methods, imaging in the same conditions and nearly at the same time points. This unique combination enables a label-free observation of processes such as morphological and position changes preceding the cell death and a follow-up fluorescence verification of cell death types in one field of view using a single instrument. In this study, PC-3 prostatic cell lines treated with plumbagin in concentrations exceeding IC50 were chosen as a model because our laboratory has a long-term experience in studying this ROS-generating agent and in characterizing this cell line [15–17]. In sum, the aim of this study was to highlight limitations of the flow-cytometry analysis of cell death and to point out advantages of MHM imaging. The hypothesis is that MHM is capable of differentiating between apoptosis and oncosis more accurately than flow-cytometry. Thus, we demonstrate a new possible application of this innovative microscopic technique. Materials and Methods Chemical and biochemical reagents Ham’s F12 medium, mycoplasma-free foetal bovine serum (FBS), penicillin/streptomycine and trypsine were purchased from PAA Laboratories GmbH (Pasching, Austria). Phosphate-buffered saline (PBS) was purchased from Invitrogen Corp. (Carlsbad, CA, USA). Ethylenediaminetetraacetic acid (EDTA), plumbagin and other chemicals of ACS purity were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), unless noted otherwise. Cell cultures Human PC-3 prostate cancer cells were used in this study. The PC-3 cell line was established from the prostatic adenocarcinoma (Grade 4) of a 62-year-old Caucasian male and derived Table 2. Classification of holographic methods. Method Optical setup Illumination source Properties Phase-shifting microscopes In-line (zero angle between object and reference beam) Low-coherence (halogen lamp, LED) + suppressed coherence noise + coherence-gating effect + lateral resolution of conventional microscopes −slow: 3 images for reconstruction Digital holographic microscopes off-axis (non-zero angle between object and reference beam) High-coherence (laser) −coherence noise −lateral resolution 2x worse than in conventional microscopes + fast: 1 image/reconstruction Coherence-controlled holographic microscopes off-axis low-coherence + suppressed coherence noise + coherence-gating effect + lateral resolution of conventional microscopes + fast: 1image/reconstruction doi:10.1371/journal.pone.0121674.t002 Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 3/16 from a metastatic site in the bones. The PC-3 cell line was purchased from HPA Culture Collections (Salisbury, UK). Cultured cell conditions The PC-3 cells were cultured in Ham’s F12 medium with 7% FBS. The medium was supplemented with penicillin (100 U/ml) and the cells were kept at 37°C in the humidified incubator with 5% CO2. Plumbagin treatment The stock solution of plumbagin was prepared in dimethylsulfoxide (DMSO) and diluted with the medium. Controls were added an equal volume of DMSO (final concentration 0.1%). The plumbagin treatment was initialized after the cells reached ~50% confluence. Previously, IC50 for plumbagin was determined as 1.50 μM using MTT. Thus, a treatment with 2 μMof plumbagin was used in this experiment to ensure the initiation of cell death. Multimodal holographic microscopy The design of the Multimodal Holographic Microscope (IPE BUT, TESCAN, Brno, Czech Republic) is based on the original concept of the Coherence-Controlled Holographic Microscope [18,19]. Holographic microscopy was initiated after 2 h of the plumbagin treatment. Time-lapse monitoring was performed for 2 h (in total 4 h of plumbagin treatment) at a frame-rate of 1 frame/min. The cells were observed in flow chambers μ-Slide I Luer Family cat. Num. 80196 (Ibidi, Martinsried, Germany) in Ham’s F12 medium. Nikon Plan 10×/0.3 and Nikon Plan Fluor 20×/0.5 objectives were used for both holographic and fluorescence observations. Interferograms for holography were taken using a CCD camera (XIMEA MR4021MC-VELETA). The fluorescence mode used a plasma light source (Sutter Instrument Lambda XL) and a CCD camera (XIMEA MR285MC-BH, 1392×1040px) was used to capture the images. Holographic and fluorescence images were collected by custom software. In fluorescence image there is no need for other image processing; however, holographic raw data have to be numerically reconstructed. The numerical reconstruction is performed by the custom software where the established methods of the fast Fourier-transform [20] and phase unwrapping [21, 22] are implemented. The output from the software is an unwrapped phase image. This image has intrinsic high contrast and can be processed by an available image processing software. We used particularly the ImageJ functions of Thresholding and Analysed Particles. At chosen time points of the time-lapse observation, the cells were segmented from the background, a threshold value for the segmentation being 0.21 rad (0.05 pg/pixel). Each cell was controlled visually, and cells in contact were separated manually. Measurements of cell surface, cell dry mass and mean cell dry mass followed. TEM visualization of ultrastructure. PC-3 cells were gently harvested by repetitive pipetting and spun down (2000 rpm, 5 min.). Briefly, the cells were fixed with 3% glutaraldehyde in a cacodylate buffer for 2 hours and washed three times for 30 minutes in 0.1 M cacodylate buffer. Then they were fixed with 0.02 M OsO4 dissolved in 0.1 M cacodylate buffer, dehydrated in alcohol, and infiltrated with acetone and No. 1 Durcuptan mixture overnight. On the following day, the cells were infiltrated with No. 2 Durcuptan mixture, embedded and polymerized. Ultrathin sections (90 nm, Ultramicrotome LKB, Bromma, Stockholm, Sweden) were transferred onto grids covered with a Formvar membrane (Marivac Ltd., Halifax, Canada). 2% uranyl acetate and Reynolds solution were used for contrast staining. The sections were viewed in the transmission electron microscope (Morgagni 268, FEI Europe B.V., Eindhoven Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 4/16 Netherlands). Software AnalySIS (Soft Imaging System, GmbH, Münster, Germany) was used for a picture analysis of the cell ultrastructure. Annexin V/propidium iodide flow-cytometry Double-staining with fluorescein isothiocyanate (FITC)/propidium iodide (PI) was performed using the annexin V-FLUOS-staining kit (Roche Applied Science) according to the manufacturer’s protocol in order to determine percentages of viable, apoptotic, and necrotic cells following the exposure to plumbagin (2 uM). Briefly, the cells were harvested by repetitive pipetting and washed two times with PBS (centrifuged at 2000 rpm for 5 min). Then they were re-suspended in 100 μl of the annexin V-FLUOS labelling solution and incubated for 15 min. in the dark at 15–25°C. Annexin V-FITC binding was detected by flow cytometry (Partec GmbH, Münster, Germany) (Ex = 488 nm, Em = 533 nm, FL1 filter for annexin V-FLUOS and FL3 filter for PI). The data were analyzed using the FloMax software version 2.5 (Partec GmbH, Münster, Germany). Results Development of the multimodal holographic microscope The employed multimodal holographic microscope was developed in cooperation between researchers from the Brno University of Technology and the TESCAN Brno Company. The MHM design (IPE BUT, TESCAN, Brno, Czech Republic) is based on the concept of the Coherence-Controlled Holographic Microscope (CCHM) described in [19]. This novel optical setup of CCHM overcomes drawbacks of the previous concept [18], while preserving all benefits and enabling multimodal imaging. The holographic mode setup is shown in Fig. 1. It is based on the Mach-Zehnder-type interferometer. The light from the source (S) passes through the collector lens (CL) and is divided by the beam splitter (BS) into two separated optical paths—object and interferometer reference arm. The beams are directed by mirrors (M). Both arms consist of condenser (C), objective (O) and tube lens (TL). In the reference arm, a diffraction grating (DG) is placed. The output lenses (OL) focus the beams onto the output plane. There the object beam and the reference beam recombine and create an interference fringes pattern (hologram), which is captured by the camera (D). The amplitude and the phase image are reconstructed numerically from the hologram. The process of the numerical reconstruction is based on the fast Fourier transform methods [20]. For time-lapse sequences, the image processing discussed in [23] is applied. The entire image reconstruction and image processing are performed by our own software. The resulting phase image can be used for classic image processing and analysis, e.g. for segmentation that defines what is the background and what is the cell in the image. From the phase image, various other visualization modalities can be easily obtained by simple numerical calculations [24]. Here we used a simulated differential interference contrast (simulated DIC) that was calculated as a 1D gradient of the quantitative phase image. Flow-cytometry analysis of cell death First, non-stained cells (control) were analyzed using flow-cytometry to set the annexin V /PI gating regions (Fig. 2A). Consequently, non-treated cells and cells treated with plumbagin for 3 h were analyzed (Fig. 3B and C, resp.). Four different phenotypes were distinguished: (a) annexin V−/PI−(lower left quadrant, Q3); estimated as viable cells; (b) annexin V+/PI−(lower right quadrant, Q4); usually estimated as apoptotic cells, but probably could contain larger Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 5/16 quantities of oncotic cells; (c) annexin V−/PI+ (upper left quadrant, Q1); fragments of damaged cells; (d) annexin V+/ PI+ (upper right quadrant, Q2); late apoptotic and necrotic cells. Compared to the non-treated cells, the fraction of annexin V+/PI−cells increased distinctly to 30.55% (compared to 4.50% in the non-treated sample), and the fraction of annexin V+/PI + increased up to 7.75% (compared to 1.70% in the non-treated sample). Furthermore, the studied cells were back-gated in the dot plot with the forward scatter/side scatter parameters of these treated cells. As a result of the back-gating, two different annexin V+/PI−populations can be seen (gated in green colour) (Fig. 2C, lower dot plot, arrows R1 and R2). Therefore, additional re-gating was performed. As a result, annexin V+/ PI−and lower FSC (region R1, supposed apoptosis) formed 9.51% of cells, and annexin V+/ PI−and higher FSC (region R2, supposed oncosis) formed 13.22% of cells (gating process not shown). Time-lapse holographic microscopy Subsequently, the same plumbagin-treated PC-3 cell line was observed using MHM. Attention was focused on changes in the cellular morphological features including cell shape, cell mass, cell spreading area, and typical structures, which could be seen in the injured cells. Using the time-lapse analysis, we identified 32% of cells which increased their volume and showed increasing blebs in the plasma membrane during the time-lapse analysis (designated as “increase size”in Fig. 2D), 24% of cells, which decreased their volume and showed apoptotic bodies (designated as “decrease size”), and 44% of cells with the unchanged volume during the 2 h of monitoring. From the phase images, we also measured cell surface mass over the time-lapse Fig 1. Holographic mode setup in Multimodal holographic microscope is based on the Mach-Zehndertype interferometer. 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. doi:10.1371/journal.pone.0121674.g001 Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 6/16 Fig 2. Differences in cell death estimation between flow cytometry and holographic microscopy. A. 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. Annexin V/PI staining, untreated cells. 92% are double negative for staining. C. 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. D. Multimodal holographic microscope, phase image. 10 × magnification 3 h after 2 μM plumbagin treatment. Red-outlined cells show size increase and oncotic phenotype, greenoutlined 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 (Table 1)E. Relative change of cell surface in individual cells (relative to initial time point). Colour coding of individual cells is based on (D). F. Mass of individual cells in pg. Note a significantly higher mass of the “decrease-size”cell population. G. Time-lapse of typical oncotic Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 7/16 observation (Fig. 2E-F). Accordingly, the surface area of the cells differed significantly between these three groups of cells, F (2, 244) = 31.05, p <0.001, namely at later time points. With regard to cell mass, the results are in accordance with the assumption that this parameter remains nearly unchanged over this short time in the cell cycle. 90% of the observed cells changed their mass by less than 7%, F(5, 244) = 0.03, p = 0.99. In contrast, the mass of cells was significantly higher in the “decrease size”group of cells as compared to the “no change”and “increase size” groups, F(5, 244) = 4.68, p = 0.01; the mass of the “decrease size”cells was on average 1.3-fold higher (Fig. 2F). By contrast to oncosis or necrosis, which are associated with the cell swelling and increased cell volume, apoptosis is connected with the loss of cell volume during the cell shrinkage (for detailed characterization of morphological changes of each cell death type see the below section of this text). Regarding the fact, 2×3 two way contingency tables were created to compare HM with the flow-cytometry results (no size change in HM vs. double-negative cells in FCM; “increase size”vs. annexin V+/PI+ population, and “decrease size”vs. annexin V+/PI−population). There was a significant difference between the assays; the proportion of the “increase size”cells was significantly higher when determined by holographic microscopy, χ 2 = 18.043, p = 0.0001 (Table 3). Because two annexin V+/PI−populations (green) were found on the FSC/SSC dot-plot by back gating, another chi-squared test was performed. Unlike in the previous testing, the “increase size”was compared with the annexin V+/PI+ population (Q2) plus R2 population (see above for details) and the “decrease size”included only the R1 gating region. Although there was still a significant difference, the proportional difference between the assays was not that profound and hence the p-level was higher (Table 3). Assessment of apoptotic, oncotic, and necrotic cells morphology Consequently, morphology of the cells after the plumbagin treatment was described using MHM, light microscopy, and verified at ultrastructural level using transmission electron microscopy (TEM). In agreement with the previous chapter, three populations of cells were observed according to size changes. Apart from cellular shrinking or swelling, other characteristics typical of distinct cell deaths were observed in MHM (see Table 1). In the oncotic cells, an intact plasma membrane with cytoplasmic blebs, nuclear chromatin clumping, and nucleus dilatation were detected. The formation of cytoplasmic blebs is well apparent in the time lapse (Fig. 2G). Some major morphological features connected with necrosis were observed too, including multiple and large cytoplasmic blebs, translucent cytoplasm, cell swelling ended by cell membrane disruption and nucleus dilatation (Fig. 3). In contrast, the shrinking group of cells exhibited characteristics typical of apoptosis: spherical shape of the cells, chromatin condensation, nuclear membrane irregularity and indeed the formation of multiple apoptotic bodies considered as an advanced stage of apoptosis (Fig. 3A, second column). Based on these characteristics, typical morphological criteria visible in MHM are illustrated in scheme (Fig. 3B). By using the fluorescence mode of MHM, we detected annexin V staining in all observed types of cell deaths (oncosis, necrosis, and apoptosis as well; Fig. 3A). In early apoptotic cells, annexin V positivity was detected, namely nearby the cell membrane (Fig. 3A, first column). All mentioned morphological characteristics were corroborated by light microscopy (data not shown) and at the ultrastructural level by using TEM. Further cell death features observable “increase size”cell indicated by red arrow in (D), simulated digital interference microscopy H. 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. doi:10.1371/journal.pone.0121674.g002 Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 8/16 Fig 3. Morphology of Apoptotic, necrotic and oncotic cells. A. 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. 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 Table 1. doi:10.1371/journal.pone.0121674.g003 Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 9/16 45. Hinson JA, Roberts DW, James LP. Mechanisms of acetaminophen-induced liver necrosis. Handb Exp Pharmacol. 2010: 369–405. doi: 10.1007/978-3-540-79088-4_17 PMID: 20020374 46. Chanan-Khan A, Srinivasan S, Czuczman MS. Prevention and management of cardiotoxicity from antineoplastic therapy. J Support Oncol. 2004; 2: 251–266. PMID: 15328825 47. Zhu JB, Okumura H, Ohtake S, Nakamura S, Nakao S. The molecular mechanism of arsenic trioxideinduced apoptosis and oncosis in leukemia/lymphoma cell lines. Acta Haematol. 2003; 110: 1–10. PMID: 12975549 Holographic Microscopy: Oncosis PLOS ONE | DOI:10.1371/journal.pone.0121674 March 24, 2015 16 / 16