Nanolive je příkladem rozvíjející se technologie v oblasti nanoskopie, která kombinuje holografii a tomografii (Tomografická difrakční mikroskopie, TDM). Tato technologie nepoužívá žádné značení, proto můžeme pozorovat buňky v jejich přirozeném prostředí, aniž bychom je jakkoliv modifikovali nebo měnili pomocí chemických značek. Můžeme tak zaznamenat všechny důležité životní buněčné pochody a tímto unikátním způsobem lépe pochopit chování buněk. Digitální holografický tomograf 3D Cell Explorer vytváří kolekci 96 vrstev v ose Z, kdy každá vrstva je vlastně detailní mapou indexů lomu (Refractive Index, RI) intracelulárních struktur. Tato inovativní technologie dokáže rekonstruovat ve vysokém rozlišení (x, y:200nm; z:400nm; t:1.7sec) kvalitní trojrozměrný obraz transparentních neznačených vzorků. Buňky je možné umístit do malého inkubátoru, a tak lze monitorovat chování buněk v kontrolovaném prostředí (teplota, CO2, 02, vlhkost) po dobu hodin, dnů i týdnů. Díky fyzikální podstatě zaznamenávání indexů lomu, je možné provádět následnou kvantitativní analýzu dat. 3D Cell Explorer -fluo obohacuje tomografická data o možnost kombinace s fluorescenčními značkami a umožňuje tak přesnou identifikaci organel, proteinů nebo dalších látek značených flourescenčně.Tato neinvazivní inovativní technika otevírá možnosti pro nové fascinující aplikace pro studium buněčného cyklu/smrti, studium infekce uvnitř buněk, lokalizaci nanočástic a imuno -onkologii.
Nanolive technology is an example of emerging smart nanoscopic technique by combining holography with tomography (Tomographic Diffractive Microscopy, TDM). This technology is 100 label free, so we can observe cells in their real environment without any behaviour modifications nor alterations due to chemical markers and capture important events all along cells life time and understanding in an unique way how cells behave. Digital Holographic Tomograph, called 3D Cell Explorer, creates a collection of 96 Z -stacked layers, each layer being a detailed Refractive Index (RI) map of the cells observed. This innovation technique guarantees three -dimensional high spatio -temporal resolution imaging (x, y:200nm; z:400nm; t:1.7sec) of transparent unlabeled specimens. Cells can be cultured and monitored under controlled environment conditions in continuous time -lapse for days/weeks using on -stage incubator. Based on the quantitative nature of the refractive index measurements, the system enables for quantitative data analysis for single cells and tissues. Moreover, 3D -Cell Explorer‑fluo enriches high quality tomographic data with fluorescent markers and enables also precise intracellular detection of organels, proteins or drugs based on fluorescence staining. This not invasive and quantitative imaging technique permits new intriguing applications in the cell death/cell cycle analysis, intracellular infection, intracellular nanoparticles localization and immuno -oncology.
K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.
- MeSH
- Cell Death drug effects radiation effects MeSH
- K562 Cells pathology drug effects radiation effects MeSH
- Time Factors MeSH
- Cytoskeleton pathology drug effects radiation effects MeSH
- DNA-Binding Proteins metabolism MeSH
- Phalloidine chemistry MeSH
- Financing, Organized MeSH
- Fluorescein-5-isothiocyanate MeSH
- Photosensitizing Agents pharmacology MeSH
- Aminolevulinic Acid pharmacology MeSH
- Humans MeSH
- Lim Kinases MeSH
- RNA, Messenger metabolism MeSH
- Microfilament Proteins metabolism MeSH
- Molecular Chaperones metabolism MeSH
- Oncogene Proteins metabolism MeSH
- Protein Kinases physiology MeSH
- HSP90 Heat-Shock Proteins metabolism MeSH
- Gene Expression Regulation MeSH
- Light MeSH
- Transcription Factors MeSH
- Carrier Proteins metabolism MeSH
- Protein-Tyrosine Kinases metabolism drug effects radiation effects MeSH
- Check Tag
- Humans MeSH
