Publikationsdatenbank

Ultra-deep tissue imaging by three-photon laser-scanning microscopy (2019)

Art

Vortrag

Autor

Niesner, Raluca (WE 2)

Kongress

29. Jahrestagung der Deutschen Gesellschaft für Zytometrie

Berlin, 25. – 27.09.2019

Quelle

Visions in Cytometry : Microscopy, Multiplexing Analysis, Flow-/Mass-Cytometry : 29th Annual Conference of the German Society for Cytometry : Abstract booklet — Deutsche Gesellschaft für Zytometrie (Hrsg.)

Berlin: DGfZ, 2019 — S. 21–22

Sprache

Englisch

Verweise

URL (Volltext): http://www.dgfz.org/blog/wp-content/uploads/2020/02/2019_DGfZ_Abstract_Booklet.pdf

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Developed three decades ago, two-photon laser-scanning microscopy led to a breakthrough in imaging cells in living organisms due to the advantages provided by the non-linear nature of two-photon excitation of fluorescence markers combined with the application of far-red and near infrared lasers. In order to achieve effective two-photon excitation, we routinely use a combination of Ti:Sa and OPO laser sources for in vivo imaging of different cells of organs of the immune system, expressing various fluorescent markers from blue to far-red and infrared fluorescence proteins. Although two-photon microscopy allows deep tissue imaging, it yields relatively poor resolution and low penetration depth when used to image through optically dense tissues such as the bone. For this reason, three-photon microscopy represents a better approach that increases the imaging depth in such tissues because of weaker scattering at higher excitation wavelengths and better contrast due to higher-order of nonlinear excitation. Here, we demonstrate three-photon imaging of different transgenic mouse organs, focusing on bone and bone marrow, and compare these results with the two-photon microscopy approach. We also focus on the great potential of label-free imaging techniques by detecting SHG and THG of different tissue structures. To enable three-photon excitation of green and red fluorescent proteins along with third-harmonic generation, a new optical parametric amplifier (OPA) at 2MHz or 1 MHz repetition rates is used. Particular interest was invested to characterize OPA laser pulses by measuring pulse widths, lateral and axial resolution. The demonstrated technique will open a new horizon in intravital deep imaging of highly scattering tissues.