Expanding Two-Photon Intravital Microscopy to the Infrared by Means of Optical Parametric Oscillator

Abstract

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.

Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinearin-vivoimaging

“…The OPO reduces photobleaching and scattering in the infrared wavelengths. [28][29][30] The OPO beam is directed toward the active region of the DMM with an incident angle of 0 deg by using appropriate polarization optics (Fig. 1).…”

The Role of CD8+ T Cells and Their Local Interaction with CD4+ T Cells in Myelin Oligodendrocyte Glycoprotein35–55–Induced Experimental Autoimmune Encephalomyelitis

“…Imaging was performed using a specialized two-photon -scanning microscope (LaVision BioTec) previously described by Herz et al (26), which allows for dual near infrared (700-1020 nm) and infrared (1050-1600 nm) excitation, that is, pulsed near infrared radiation is generated by an automatically tunable Ti:Sa laser, 10% of which is coupled into a scan head. Ninety percent of Ti:Sa laser power is coupled into a synchronously pumped optical parametric oscillator.…”

When multiphoton microscopy sees near infrared

“…Several studies clearly demonstrate the benefits of NIR light for deep and vitalpenetration and the spatial resolution imaging. NIR, usually in a range between 1050 and 1300 nm, increases by 40-80% the maximal light penetration and the spatial resolution at 70 mm depth is improved by 1.7 fold in heavily scattering samples such as brain and cortex slices, compared to conventional Ti:Sapphire excitation [2,3]. Furthermore, NIR excitation increases the signal to noise ratio by lowering the autofluorescence background usually found when using visible light.…”