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Over the past decade the range of available excitation wavelengths used in nonlinear microscopy has continuously extended within the near infrared window. Nowadays, excitation wavelengths ranging from 750 nm to 1300 nm are routinely used to perform multi-parametric imaging [1,2] and multiple wavelength excitation are used in many techniques, including multicolor 2-photon excited fluorescence imaging, Coherent Anti-stokes Raman Scattering (CARS), Stimulated Raman Scattering (SRS) or sum-frequency generation. While this trend opens new perspectives and applications in the biomedical sciences, it also raises new technical issues. For instance, it calls for new standards for quantifying and comparing the performances of nonlinear microscopes over a broad range of wavelengths. In particular, microscopes equipped with multiple femtosecond sources spanning the entire near-infrared wavelength range are often problematic to characterize with current approaches based on fluorescent probes.
In this study, we present a new and straightforward method to quantify the imaging properties of nonlinear microscopes over a broad range of excitation wavelengths [3]. We show that harmonic generation nanoprobes are a unique tool to map the spatial resolution, field curvature and chromatic aberrations of nonlinear microscopes with a precision below the diffraction limit, across the whole field of view, and with a single calibration sample. We analyze and compare measurements obtained with several microscope objectives designed for multiphoton microscopy over the 850-1100nm wavelength range. Finally, we discuss strategies to minimize the impact of chromatic aberrations during multicolor imaging and we show how our metrology can be used for the post-acquisition correction of chromatic aberrations.
[1] Mahou et al. Nat. Methods 9, 815-18 (2012).
[2] Alexander et al. Curr. Opin. Cell Biol. 25 , 659-71 (2013)
[3] Mahou et al., submitted.
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