Development of a multiphoton-multicolor and super-resolution STED microscope for in vivo experiments

Two-photon excitation (2PE) microscopy [1] has proven to be an excellent technique for in vivo fluorescence imaging of deep structures and tissues due to low absorption and scattering of infrared light. The combination of two laser beams introduces the possibility of further imaging capabilities, such as simultaneous excitation of three fluorophores (multiphoton-multicolor [2]) and sub-diffraction resolution of cellular structures (STimulated Emission Depletion, STED [3,4,5,6,7]). Here we present a cost-effective solution that combines both techniques by use of a homemade optical bench equipped with a pulsed laser split in two beams by an Optical Parametric Oscillator (OPO). Preliminary experiments were successfully performed with the multiphoton-multicolor technique, setting proper conditions to perform experiments for the study of neutrophil mobilization in skull bone marrow of diabetic mice. In the STED configuration, we successfully tested the fluorescence depletion effect and the optical generation of a doughnut-shaped point-spread-function by the depletion laser, both conditions required to obtain super-resolution. A preliminary super-resolution performance was obtained by visualizing the outer mitochondrial membrane stained with Alexa 647 dye.