Live Cell Imaging
Advanced Bioimaging Modalities
Advanced Microscopy Techniques for continuous observation of living cells
The objectives of the project are to utilize minimally invasive, high resolution, four dimensions fs laser raster-scanning imaging and analysis methods through the systematic use of multiphoton excited fluorescence (MPEF), polarization sensitive second harmonic generation (P-SHG) and third harmonic generation (THG), to quantitatively characterize the structure and function of living cells and tissues at video rates.
Critical insight and characterization of the fundamental nature of cellular and tissue function traditionally relies on detailed morphological information at the microscopic level. In recent years minimally invasive laser raster-scanning imaging techniques like MPEF (two-photon and three-photon), P-SHG and THG have emerged as new powerful high resolution optical modalities for quantitative characterization of biological samples.
Both the above advanced optical microscopy techniques are based on minimally invasive fs laser irradiation and provide intrinsic z-sectioning due to signals nonlinear dependence on the excitation photon flux. Since the fs laser interaction is minimal, fairly nonlinear microscopy is considered as the most appropriate for live imaging. It is based on signals originating from endogenous non-centrosymmeric molecular assemblies or on signals from exogenous species like fluorescence chromophores or nanoparticles to provide contrast. Furthermore, the infrared wavelengths used allow penetration depths (of several hundred microns into highly turbid tissues), unreachable by the common linear fluorescence or confocal microscopy.
Endogenous SHG arises from a highly organized assembly of non-centrosymmetric biomolecules (harmonophores), while exogenous SHG arises from SHG active nanoparticles or from well-ordered non-linear dyes. SHG imaging provides data about the organization and structural symmetries of SHG active supramolecular assemblies in cells and tissues, while TPEF supplies information about the nature and the concentration of fluorophores. In other words, SHG provides structural information while TPEF provides molecular information.
The hypothesis that we are testing with our (polarization and nano-surgery) microscope is that the differences in the orientations of the harmonophores and the concentrations of the fluorophores will serve as quantitative imaging biomarkers and will provide the means for quantitative evaluation of plethora interdisciplinary research scenarios, which require live cell imaging in order to be studied.
Figure 1: TPEF imaging in living neurons.
Figure 2: 2p-Calcium Imaging of neural aggregates.
- “Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy” S. Bancelin, B. Lynch, C. Bonod-Bidaud, G. Ducourthial, S. Psilodimitrakopoulos, P. Dokladal, J.-M. Allain, M.-C. Schanne-Klein, and F. Ruggiero, Scientific Reports 5, 17635 (2015).
- “Monitoring myosin conformational fast changes in-vivo with instantaneous single scan polarization-SHG microscopy” S. Psilodimitrakopoulos, D. Artigas, and P. Loza-Alvarez, Biomedical Opt. Express, 5, 4362 (2014).
- “Quantitative imaging of microtubule alteration as an early marker of axonal degeneration after ischemia in neurons” S. Psilodimitrakopoulos, V. Petegnief, N. de Vera, O. Hernandez, D. Artigas, A. M. Planas, and P. Loza-Alvarez, Biophys. J., 104, 968 (2013).
- “Femtosecond laser axotomy in Caenorhabditis elegans, and collateral damage assessment using a combination of linear and nonlinear imaging techniques” S. I.C.O. Santos, M. Mathew, O. E. Olarte, S. Psilodimitrakopoulos, and Pablo Loza-Alvarez, PLoS ONE, 8, e58600 (2013).
- “In-depth analysis of egg-tempera paint layers by multiphoton excitation fluorescence microscopy”, Sustainability 12:3831 (2020). Alice Dal Fovo, Mikel Sanz, Mohamed Oujja, Raffaella Fontana, Sar, Mattana, Riccardo Cicchi, Piotr Targowski, Marcin Sylwestrzak, Aldo Romani, Chiara Grazia, George Filippidis, Sotiris Psilodimitrakopoulos, Andreas Lemonis, Marta Castillejo
- “Neural Stem Cell Delivery via Porous Collagen Scaffolds Promotes Neuronal Differentiation and Locomotion Recovery in Spinal Cord Injury“, Nature Regenerative Medicine, 5, 12 (2020) Alexandra Kourgiantaki, Dimitrios Tzeranis, Kanelina Karali, Sotirios Psilodimitrakopoulos, Konstantina Georgelou, Ioannis Yannas, Emmanuel Stratakis, Kyriaki Sidiropoulou, Ioannis Charalampopoulos, Efstathia Bampoula, and Achille Gravanis
- “Three-dimensional characterization of collagen remodeling in cell-seeded collagen scaffolds via polarization second harmonic generation”,Dionysios Xydias, Georgios Ziakas, Sotiris Psilodimitrakopoulos, Andreas Lemonis, Eleni Bagli, Theodore Fotsis, Achille Gravanis, Dimitrios S. Tzeranis, and Emmanuel Stratakis, Biomed. Opt. Express 12, 1136-1153, 2021
Dr. Emmanuel Stratakis
Dr. Sotiris Psilodimitrakopoulos
Mr. Dionysios Xydias
Mrs. Maria Kefalogianni
Dr. Lina Papadimitriou
Mrs. Rodoula Zafeiri
Mr. Andreas Lemonis
Prof. Dimitris Tzeranis
Prof. Kyriaki Sidiropoulou
Dr. Katerina Gkirtzimanaki