Harmonic nanoparticles for regenerative research.
In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength. Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva.
Flavio
F
Ronzoni
Physics Department, GAP-Biophotonics, University of Geneva.
Thibaud
T
Magouroux
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva.
Remi
R
Vernet
Laboratoire d'Optique Biomédicale (LOB), Faculté des Sciences et Techniques de l'Ingénieur, École Polytechnique Fédérale de Lausanne.
Jérôme
J
Extermann
Department of Clinical Medicine, School of Medicine, Trinity College Dublin.
Darragh
D
Crotty
School of Medicine and CRANN, Trinity College Dublin.
Adriele
A
Prina-Mello
Nikon AG Instruments.
Daniel
D
Ciepielewski
Department of Clinical Medicine, School of Medicine, Trinity College Dublin.
Yuri
Y
Volkov
Physics Department, GAP-Biophotonics, University of Geneva; luigi.bonacina@unige.ch.
Luigi
L
Bonacina
Physics Department, GAP-Biophotonics, University of Geneva.
Jean-Pierre
JP
Wolf
Department of Pathology and Immunology, Faculty of Medicine, University of Geneva.
Marisa
M
Jaconi
D002450:Cell Communication; D002454:Cell Differentiation / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D032383:Myocytes, Cardiac / Q000166:cytology; D053758:Nanoparticles / Q000737:chemistry*; D009556:Niobium / Q000737:chemistry; D010087:Oxides / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D012038:Regeneration / Q000502:physiology*; D044968:Regenerative Medicine
D010087:Oxides; D009556:Niobium; C477466:potassium niobate; D011188:Potassium