Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates.
We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging.
1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101.
2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53–54.
Ingo Gregor, Robert Ros, and Jörg Enderlein, "Non-linear image scanning microscopy (Conference Presentation)," Proc. SPIE 10071, Single Molecule Spectroscopy and Superresolution Imaging X, 100710C (Presented at SPIE BiOS: January 28, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2255891.5380386346001.
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