# Line-Shaped Illumination: A Promising Configuration for a Flexible Two-Photon Microscopy Setup
## 2 Description of the Experimental Setup  
The idea behind the proposed microscope setup arises from an exhaustive review of the scientific literature about line-scanning microscopes, in order to investigate the worsening of the resolution in comparison to point-scanning systems in detail. The reason is usually ascribed to the weak focusing, which occurs in only one of the two transverse  
Figure 1: Adapted from [11]: ideal scheme of the considered problem. directions rather than to the astigmatism due to the cylindrical optics used for beam shaping. However, according to numerical simulations that we have performed, we have come to the conclusion that the role of such astigmatism is severely underestimated. Indeed, if the two focal volumes generated by the microscope objective both lie inside the sample, two contributions of nonlinear fluorescence to the detected signal will be generated, at two different depths, thus leading to a degradation of the axial resolution. Moreover, if the separation between the two focal planes is small, the intensity of the excitation beam can be high enough to generate nonlinear fluorescence in all the regions in between. The microscope setup has then been designed so that the focal distance of one of the two planes is made infinite by collimating the beam out of the microscope objective in one of the two transverse directions. In such a way, the sectioning capability of the microscope is expected to be preserved, notwithstanding the linear focusing. This is possible by means of a proper arrangement of the lenses used in the optical system, which has not been considered in previous works [12; 13]. As for the excitation intensity along the focal line, a uniform light distribution is highly desirable in order to yield a high in-plane resolution. In fact, if the intensity varies along the line, the power level being necessary to excite a detectable amount of nonlinear fluorescence in the low-intensity regions could possibly saturate the nonlinear absorption in the high-intensity regions, thus worsening the transverse resolution. As the beam emitted by a typical excitation laser source has a Gaussian shape, common cylindrical lenses generate a bell-shaped intensity distribution along the focal line. In order to avoid that, the most straightforward solution is the overfilling of the entrance pupil of the objective, so that the apodization of the beam will limit the intensity variations. Besides the residual unbalance in the intensity distribution, this approach would clearly suffer from an inefficient use of the laser power. A more efficient and better-performing solution can be obtained by means of a Powell lens, which is able to convert a Gaussian beam into a flat-top rectangular intensity distribution by farning out an input collimated beam in one dimension. The design of the microscope turns out to be more complex, but the benefit in terms of performance appears to be appealing. The excitation light source employed in the development of the LSTPM setup is a continuous-wave, mode-locked Ti:Sapphire laser (Tsunami, Spectra Physics), emitting femtosecond pulses at the repetition rate of 82 MHz. The beam has a full-width half-maximum diameter of 2 mm and is magnified by a factor of 1.5 by a Keplerian telescope before entering the microscope setup, which is shown in Figure 2. As the beam profile is Gaussian, a Powell lens with a fun angle of 20\({}^{\circ}\) is used in order to yield a flat-top profile in one of the two transverse directions. In such direction, the beam turns out to be highly divergent and it is subsequently recollimated by means of a 25 mm focal length cylindrical lens, thus making up an effective Galilean expander. Figure 2: Microscope setup. The beam then impinges on a single-axis galvanometer scan mirror providing deflection along the perpendicular direction at a maximum angle of 25\({}^{\circ}\).