Nanoscopy in a living multicellular organism expressing GFP.

We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556-592 nm.

STED microscopy with a MHz pulsed stimulated-Raman-scattering source.

We perform stimulated emission depletion (STED) microscopy with a novel light source consisting of a fiber-amplified, frequency doubled laser operating with a 1 MHz repetition rate and a 530 nm output coupled into a standard single mode fiber to produce a tunable spectrum of discrete peaks via stimulated Raman scattering (SRS). Using peaks at 585, 600, and 616 nm as STED light we perform STED microscopy with resolution down to 20-30 nm. The nanosecond pulsed light source should prove valuable for all forms of microscopy requiring both brilliance and multiple wavelengths in the visible range.

Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source.

We describe a subdiffraction-resolution far-field fluorescence microscope employing stimulated emission depletion (STED) with a light source consisting of a microchip laser coupled into a standard single-mode fiber, which, via stimulated Raman scattering (SRS), yields a comb-like spectrum of seven discrete peaks extending from the fundamental wavelength at 532 nm to 620 nm. Each of the spectral peaks can be used as STED light for overcoming the diffraction barrier. This SRS light source enables the simple implementation of multicolor STED and provides a spectral output with multiple available wavelengths from green to red with potential for further expansion.