Stimulated emission depletion microscopy with stimulated Raman scattering light sources.

Abstract

Light sources offering a spectrally flexible output in the yellow-orange region of the visible spectrum are of great interest for stimulated emission depletion (STED) microscopy because they enable super-resolution imaging of the fluorescent proteins which emit in this range, in particular the green and yellow fluorescent proteins (GFP and YFP) and their variants. Lasers exist which produce yellow-orange light, but each has distinct disadvantages for use in a STED microscope. Titanium:sapphire lasers pumping optical parametric oscillators are used, but are technically complex and often require careful adjustment. Single-color fiber lasers are an option but do not offer valuable spectral tunability. Supercontinuum fiber lasers, due to the fact that they generate broad continua, have low pump conversion efficiencies to narrow wavelength ranges within their output spectrum, ultimately limiting the repetition rate and output power in a narrow spectral range. This thesis presents a novel light source for STED microscopy based on stimulated Raman scattering (SRS) in standard optical fiber. The source produces pulsed light of high intensity from the green to the red range of the visible spectrum, and can be used to easily attain resolutions of tens of nanometers in a STED image. The physical principles of the SRS light source are discussed. The implementation of three versions with increasing STED imaging performance is demonstrated, with repetition rates increasing from tens of kHz to 20 MHz. With the 20 MHz SRS source beam scanning was incorporated into the STED microscope setup and used for sub-diffraction imaging of living cells expressing GFP- and YFP-fusion proteins. To date the SRS light source provides the best option for sub-diffraction STED imaging with GFP.