Superresolved CARS by coherent image scanning
Superresolution microscopy has transformed many life science imaging applications. Yet, a similar advance towards breaking the resolution barrier in coherent nonlinear microscopy is yet to occur. Here we present the implementation of image scanning microscopy (ISM) to coherent anti-Stokes Raman imaging, showing a significant resolution enhancement. In ISM, a pixelated detector serves as the pinhole in confocal microscopy, whereby the excitation spot is magnified such that every pixel acts as a small pinhole. Signals collected from multiple pixels are then analysed to generate a higher resolution image. The difficulty in applying ISM to coherent imaging is that the analysis process requires exact knowledge not only of the amplitude of the signal but also its phase. We implement CARS-ISM using nearly inline interferometry, whereby a reference signal is generated in a glass slide and is directed towards the sample along with the pump and stokes beams. Dispersion compensation and phase stepping of the reference beam is done in a 4f pulse shaper inserted between the generation of the reference and the sample. Using this system, we obtain superresolved CARS images in the lipid C-H stretch band when using pump and Stokes beams generated by a Ti:Sapphire laser and a synchronously pumped OPO. Importantly, the phase of the CARS signal is spatially variant due to the different rations of resonant and nonresonant contributions. To further characterize the resolution enhancement we use polymer air-force like grating targets which show that the use of ISM can lead to a ~1.5 times better resolution even without advanced image processing, and holds the potential for further resolution increase. The potential of this technique as a general-purpose booster of resolution in coherent nonlinear microscopy and possible implementations in epi-detection mode will also be discussed.