Nonlinear microscopy (NLM) has several inherent advantages in contrast with confocal laser scanning microscopy. These are intrinsic optical sectioning, larger penetration depths, reduced photo damage etc. This has allowed the technique to become an important tool in biology [1
]. These advantages rely on the fact that NLM is based on a multi-photon effect where the generated signals are nonlinearly dependent on the excitation intensity. This can be achieved when photons of ultrashort pulses are tightly focused through a high Numerical Aperture (NA) microscope objective resulting in a confined focal excitation volume [1
]. Although this technique allows larger penetration depths compared with confocal microscopy, aberration effects and scattering limit the full exploitation of such capability. Aberrations lead to a spreading of the focusing spot inside the sample, both in axial and lateral directions, reducing the resolution and the intensity. As a consequence, the contrast of the imaged sample drops drastically [3
]. Aberrations in a microscope are not only caused by the intrinsic optical elements quality and alignment, but also by the refractive index mismatch from the objective, immersion media, cover glass, and more importantly, by the inhomogeneous structure of biological samples [6
]. In addition, when imaging inside semi-transparent samples, the image quality is weakly affected by scattering up to a depth of several scattering lengths [7
To deal with these problems, microscopists are starting to adopt different Adaptive Optics (AO) sensing schemes to correct the aberrated wavefronts (WF). Such WF can be corrected in the excitation beam [8
], in the collected signal beam or in both. For example, in wide field techniques such as PALM or STORM, only the correction of the collected signal is important in order to maintain the contrast of the acquired images [9
]. In confocal microscopy, both the excitation and collected beams need to be compensated to improve the intensity of the reconstructed images [10
]. In NLM, there is no need to correct on the collected beam as no confocal aperture is required. However, correcting the excitation beam is a must as this will ensure a better focusing and therefore, a more efficient nonlinear (NL) process.
In practice, measuring and correcting excitation beam aberrations at the sample plane of a nonlinear microscope is a difficult task. Nevertheless, some strategies to do this have already been implemented. These strategies can be divided into sensor-based and sensor-less techniques.
In sensor-less schemes, iterative algorithms are generally used to control a deformable mirror (DM) or a Spatial Light Modulator [12
]. This compensates the unknown existing optical aberrations without having to measure them. Although effective, optimization is performed by improving a merit function based on an image-quality related parameter, such as the total intensity within the imaged area [14
]. The success of the algorithm is dependent on the initial guess of the optimization process, the employed algorithm, (genetic, modal, etc.) and the stopping criteria. Although it has been reported the use of optimized algorithms that reduce sample exposure [7
], this still implies that an area of interest within the sample has to be exposed a considerable number of times. This may result in unwanted exposure which is prone to produce photo-bleaching and photo-toxic effects on the sample [17
In sensor-based schemes, the correction can be performed by measuring the optical aberrations through a sensing device such as a wavefront sensor (WFS) or an interferometer [6
]; then the information is then fed into an adaptive element. The standard implementation of this technique, when used for microscopy applications, suffers from important drawbacks. Collecting the excitation beam in forward direction requires careful consideration: first, because the upper layers of the sample will produce aberrations that might interfere with the measurement if the lower layers are being corrected, and second because an additional lens with the same or higher numerical aperture than the used microscope objective must be introduced into the setup. In this case, the optical quality and alignment of this element with the optical axis induces undesirable aberrations that must be calibrated. In a backward configuration the backscattered excitation signal (normally collected in double pass scheme) has the same intensity level as the spurious reflections, making it difficult to distinguish between these two [11
Despite that, sensor based schemes are powerful techniques as WF aberrations are directly obtained. Thus, alternative solutions can be considered. Interferometric methods or confocal depth selection mechanisms have been successfully applied for a direct WF measurement. These principles have proved to work well as they enable to filter/select the excitation beam at the imaged focal plane. However, they work only in thick and highly scattering samples [11
] or rely on complex experimental setups [19
Given this, other methodologies, similar to those already used in astronomy, have been used. In particular AO has been broadly used in astronomy to compensate for the changing turbulent medium in the atmosphere which produces aberrations and deteriorates the image of objects to be observed. By employing a WFS it is possible to measure the distortions produced when distant light sources travel through the changing atmosphere, employing a reference point source called guide star. This can be a natural star or a laser producing fluorescence in the atmosphere [21
]. Then such information is fed to a DM, in order to restore the image quality.
The above principle has been applied for moderate scattering samples imaged through linear fluorescence microscopy. This method consists of artificially inserting a small secondary light source (i.e. a fluorescent bead) inside the sample [22
] which is fixed after its preparation. This allows for a direct measurement of the sample aberrations using a Shack-Hartmann (SH) WFS, therefore allowing the correction of the excitation beam aberrations. However, since this technique is based on the introduction of beads inside the sample by using a microinjection needle or negative pressure protocols [22
], it is prone to cause sample damage, limiting its potential for in vivo
imaging studies. Besides, this, beads are randomly distributed inside the fixed sample, therefore a suitable bead placed at the right location should be found in the field of view (FOV) and for each depth to be imaged. This adds complexity to the sample preparation and to the aberration measurement process.
In order to extend the guide star concept for microscopy applications, a key point not properly appreciated before is that fluorescence, being an incoherent process, does not contain information about the aberrations gained by the excitation beam. Therefore, it allows for the implementation of single pass aberration measurement schemes.
Two-photon excited fluorescence (TPEF) naturally produces a small confined volume which can be used as an incoherent secondary light source. Thus in this work we show how this point source, which we refer to as the ‘nonlinear guide-star’ (NL-GS), can be used in a practical and noninvasive way for single pass measurements of sample and objective aberrations in NLM using a SH WFS sensor. Furthermore, we also show that this information can be used for correcting such aberrations in a single step with the help of a DM. This is demonstrated using both fixed and in vivo biological samples in which the enhancement of the corrected images, when compared with the non-corrected ones, results in an improvement of more than one order of magnitude in the total collected signal intensity.