The Stratakis Lab

The Stratakis Lab

Ultrafast Laser Micro and Nano Processing Laboratory

Live Cell Imaging

Live Cell Imaging

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 scanning imaging and analysis methods through the systematic use of multiphoton excited fluorescence (MPEF) and polarization sensitive second harmonic generation (P-SHG), to quantitatively characterize the structure and function of living cells and tissues at video rates.

Contact Person(s):
Dr. Emmanuel Stratakis

Dr. Sotiris Psilodimitrakopoulos

 

Research Topics

Multiphoton Microscopy

 

Abstract

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 scanning imaging techniques, like multi-photon excited fluorescence (MPEF) and polarization sensitive second harmonic generation (P-SHG), 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 quadratic dependence on the excitation photon flux. Since the fs laser interaction is minimal, fairly both optical microscopies are considered as the most appropriate for live imaging. They are based on signals from endogenous non-centrosymmeric molecular assemblies or on 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 fluorescence or confocal laser scanning microscopies.
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.

In our lab, live cell imaging is used to characterize cell-to-cell and cell-to-material interactions, in terms of adhesion, proliferation, migration, growth, differentiation and networking using cell lines and primary cells, in environments like the laser engineered scaffolds described in RT3 and RT4. Moreover, live cell imaging is exercised in testing biocompatibility of fs laser micro- and nano- processed materials, as well as in characterizing fs laser engineered tissues. For example, collagen based engineered scaffolds are characterized by means of P-SHG and the so-called anisotropy parameter B. Finally, SHG active nanoparticles and nanorods are utilized as novel imaging probes.

Figure: MPEF imaging x-axis collage of Living GFP transfected N2a cells. Scale bar on lower right shows 0.5mm. 3D-Collage imaging can be extended to cover big areas, of the order of centimeters.

Figure: 3D-SHG microscopy of fs engineered micro-channels in dried collagen gel

Figure: TPEF imaging in neurons.

 

Figure: TPEF imaging in neurons.

Figure: TPEF imaging in neurons.

 

 

 

Collaborators

Prof. Achilleas Gravanis
Dr. Dimitris Tzeranis

 

Publications

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).

⦁ Molecular engineering of chromophores for combined second-harmonic and two-photon fluorescence in cellular imaging
E. Meulenaere, W.-Q. Chen, S. V. Cleuvenbergen, M.-L. Zheng, S. Psilodimitrakopoulos, R. Paesen, J.-M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez, X.-M. Duan, and K. Clays, Chem. Sci., 3, 984 (2012).

⦁ Ultrastructural analysis of myocardiocyte sarcomeric changes in relation with cardiac dysfunction in human fetuses with intrauterine growth restriction
J. I Iruretagoyena, I. Torre, I. Amat-Roldan, S. Psilodimitrakopoulos, F. Crispi, P. Garcia-Canadilla, A. Gonzalez-Tendero, A Nadal, E. Eixarch, P. Loza-Alvarez, D. Artigas, and E. Gratacos, AJOG, 204, S34 (2011).

⦁ Estimation of the effective orientation of the SHG source in primary cortical neurons
S. Psilodimitrakopoulos, V. Petegnief, G. Soria, I. Amat-Roldan, D. Artigas, A. M. Planas, and P Loza-Alvarez, Opt. Express, 17, 14418 (2009).

⦁ Quantitative discrimination between endogenous SHG sources in mammalian tissue, based on their polarization response
S. Psilodimitrakopoulos, D. Artigas, G. Soria, I. Amat-Roldan, A. M. Planas, and P. Loza-Alvarez, Opt. Express, 17, 10168 (2009).

⦁ In vivo, pixel resolution mapping of thick Filaments’ orientation in non-fibrilar muscle using polarization sensitive second harmonic generation microscopy
S. Psilodimitrakopoulos, S. Santos, I. Amat-Roldan, A. Thayil K. N, D. Artigas, and P. Loza-Alvarez, J. Biomed. Opt., 14, 014001 (2009).

 

 

Project Members
Dr. Emmanuel Stratakis
Dr. Sotiris Psilodimitrakopoulos
Dr. Paraskevi Kavatzikidou
Dr. Kanelina karali
Ms. Eleftheria Babaliari
Ms. Christina Lanara
Mr. Andreas Lemonis

Back