PCF infrared continuum for multiwavelength two photon microscopy

Thanks to the non linearity arising when femtosecond pulses are coupled into Photonic Crystal Fibers (PCFs), continuum spectra can be generated starting from single wavelength lasers. Multiphoton microscopy [1], fluorescence lifetime imaging [2], stimulated emission depletion microscopy [3], and optical coherence tomography [4] have been previously realized using such kind of excitation source, covering most of the state-of-the-art biological imaging techniques. Usually the properties of PCFs in terms of anomalous dispersion and high non linearity are exploited to produce the greater spectral broadening. We choose instead to pump a PCF with a selected dispersion profile in the normal dispersion region. This approach results in effects: reduction of non linearity, flat spectrum at the output and reduced amplitude noise in the different spectral bands generated [5]. We characterize the imaging performances of the system mainly in two ways. First of all by measuring the Point Spread Function (PSF) using sub-resolution fluorescent beads: sub micron radial resolution and micron optical sectioning is achieved in the whole spectrum. Most important, we compared the images obtained with a 30nm wide band selected from the continuum around 780nm with the ones using a single wavelength 785nm source (see Fig.1): Signal to Noise Ratio (SNR) and image quality are comparable, thus demonstrating the validity of our approach. The spectrum at the output of the fiber can then be arbitrarily shaped to select the desired excitation wavelength in the range from 700nm to 1000nm, where the two photon cross sections of the most common fluorophores are peaked.