We developed a new class of two-photon excitation–stimulated emission depletion (2PE-STED) optical microscope. In this work, we show the opportunity to perform superresolved fluorescence imaging, exciting and stimulating the emission of a fluorophore by means of a single wavelength. We show that a widely used red-emitting fluorophore, ATTO647N, can be two-photon excited at a wavelength allowing both 2PE and STED using the very same laser source. This fact opens the possibility to perform 2PE microscopy at four to five times STED-improved resolution, while exploiting the intrinsic advantages of nonlinear excitation.
Two-photon excitation (2PE) fluorescence microscopy is a widely used technique in medicine and biology. Its characteristics make it particularly suitable for deep tissue and in vivo imaging applications. The use of infrared wavelengths guarantees a high penetration depth in scattering samples and a reduced photodamage in living specimens. Moreover, the quadratic dependence of the excitation allows intrinsical rejection of the out-of-focus fluorescence background and reduces the FWHM of the focal spot by approximately √2 compared to the diffraction limited spot of excitation light. However, 2PE cannot be considered a genuine superresolution technique: The "doubling" in wavelength causes that the size of the diffraction spot of excitation light is twice than the one achievable by single-photon excitation (1PE). In 1994, Hell and Wichmann introduced the stimulated emission depletion (STED) concept as a way to achieve optical superresolution by breaking the diffraction barrier. In general, STED imaging is based on the fluorescence switching due to stimulated emission depletion introduced by a second red shifted beam. The fluorescent sample is probed by a focused excitation beam overlaid with a STED beam commonly shaped to a doughnut-like intensity distribution featuring zero intensity in the center. Because of the saturation of the stimulated emission depletion, the fluorescence is switched in the whole focal area except for the small region around the zero of the STED beam. The resulting resolution is theoretically unlimited being governed by the efficiency of the depletion process. Recently, STED has been proposed coupled with 2PE microscopy combining the advantages of 2PE with its superresolution ability. In general, continuous wave (CW)-STED is preferred for 2PE imaging due to a simplification of the experimental setup at the expenses of an increased cost. Unfortunately, the use of two distinct wavelengths for excitation and depletion requires mostly a special optical filter design making the setup invariable in terms of the choice of the marker dye, and potential light beam distortions have to be treated separately. Working with only just one wavelength for both excitation and STED, one directly simplifies the image formation scheme. In this paper, we propose a method to perform 2PE-STED imaging using a single wavelength (SW) and, consequently, the very same laser source for 2PE and depletion. Such a SW approach is in agreement with early reported spectral evidences for different dyes.