Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator.
Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.
Herz
Josephine
J
Cecilie Vogt Klinik, Charité-University Medicine Berlin, Berlin, Germany.
Siffrin
Volker
V
Hauser
Anja E
AE
Brandt
Alexander U
AU
Leuenberger
Tina
T
Radbruch
Helena
H
Zipp
Frauke
F
Niesner
Raluca A
RA
D000537:Aluminum Oxide; D000818:Animals; D002460:Cell Line; D003116:Color; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007259:Infrared Rays; D007834:Lasers; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008853:Microscopy / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D055620:Optical Phenomena; D038761:Photobleaching; D017785:Photons; D014025:Titanium; D000097573:Red Fluorescent Protein
D000537:Aluminum Oxide; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D014025:Titanium; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein