@conference{
author = "Isaković, Anđelka and Stanojević, Željka and Zogović, Nevena and Jovanić, Svetlana and Rabasović, Mihajlo and Krmpot, Aleksandar and Pantelić, Dejan and Misirlić-Denčić, Sonja",
year = "2015",
abstract = "Apoptosis, or programmed cell death type I, is a process in which sequence of events leads to degradation of cell content, cell shrinkage, membrane changes and fragmentation of nucleus. In the execution phase of apoptosis, breaking down of cell cytoskeleton causes the cell membrane to bulge outward - phenomenon known as membrane blebbing. The end result is formation of apoptotic bodies, membrane-bound vesicles containing organelles, sometimes with nuclear fragments. Induction of apoptosis is considered as best approach in anticancer therapy with cytotoxic drugs [1]. Therefore, detecting morphological features of tumor cells under the influence of cytotoxic agents is of great scientific importance. One of the obstacles is to visualize and analyze morphological changes in living suspension cells (e.g. leukemic cells) using conventional light and/or fluorescence microscopy. Therefore, transmission electron microscopy is often used. Although this gives deep insight into subcellular morphology and changes, methodology employed is often time-consuming, expensive and involves usage of multiple toxic substances. Given all of the above, we analyzed apoptotic changes in commercial human acute promyelocytic leukemic (HL-60) cell line treated with well-known apoptosis- inducing antitumor agent cisplatin [2], using second-harmonic generation (SHG) imaging, using femtosecond laser microscopy homemade nonlinear laser scanning microscope [3] in Two Photon Excitation Fluorescence (TPEF) mode. Cells were grown under standard conditions [4], seeded in 12-well plates, and treated with cisplatin for 48h in the IC50 value concentration range (the IC50 value is a concentration that decreases cell viability for 50 %, compared to untreated cells). After the treatment, cells were incubated in dark with supravital fluorescent dye acridine orange (AO) for 30 minutes. The ≈10 μl drop of cells was then placed on microscope slides and covered with glass cover slips. Control (untreated cells) and cells treated with cisplatin were visualized and photographed using TPEF SHG imaging. Sections of cells 1,6 μm thick were made, and 3D image reconstruction was performed. Untreated cells were seen as round, with intact cell membrane and clearly visible nucleus and nucleolus, as expected. On the other hand, leukemic cells treated with cisplatin showed changes in the morphology. They were smaller in diameter, and cell nucleus was fragmented. Furthermore, cell membrane changes were also seen, numerous protrusions, suggesting membrane blebbing and initial phases of apoptotic bodies formation. 3D image reconstruction further confirmed observed morphological cell changes typical for apoptosis. Performed experiments and obtained results suggest that SHG TPEF imaging could be used for the detection of morphology phenomena related to cell apoptosis, which can be of importance in cancer research, especially in the area of understanding cytotoxic mechanism of action of novel potential antileukemic agents.",
publisher = "Belgrade : Vinča Institute of Nuclear Sciences",
journal = "Book of Abstracts: The Fifth international school and conference on photonics & COST actions: MP1204, BM1205 and MP1205 & the Second international workshop: Control of light and matter waves propagation and localization in photonic lattices: Photonica 2015; 2015 Aug 24-28; Belgrade, Serbia",
title = "Apoptotic changes visualization in cisplatin-treated leukemic cells using second-harmonic generation imaging",
pages = "140-141",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6775"
}
