Abstract
In laser-scanning microscopy often an off-the-shelf achromatic doublet is used as a scan lens which can reduce the available diffraction-limited field-of-view (FOV) by a factor of 3 and introduce chromatic aberrations that are scan angle dependent. Here we present several simple lens designs of superior quality that fully make use of high-NA low-magnification objectives, offering diffraction-limited imaging over a large FOV and wavelength range. We constructed a two-photon laser-scanning microscope with optimized custom lenses which had a near diffraction limit point-spread-function (PSF) with less than 3.6% variation over a 400 µm FOV and less than 0.5 µm lateral color between 750 and 1050 nm.
Highlights
Laser-scanning two-photon microscopy [1] has revolutionized the life sciences
In a laser-scanning microscope (Fig. 1), scanning is achieved by placing the rotation axis of a galvanometric mirror at the back-focal plane of a scan lens
The scan lens together with the tube lens project an enlarged image of the laser beam from the galvanometric mirror to the back-focal plane (BFP) such that the centers of the beam at both conjugate planes remain stationary while scanning the sample
Summary
Laser-scanning two-photon microscopy [1] has revolutionized the life sciences. It enabled the acquisition of fluorescent images from depths greater than 500 μm within light-scattering tissues [2,3,4] with an optical resolution comparable to confocal microscopy [5] due to the nonlinear process of two-photon absorption which confines the excitation volume [3]. As we will show, such lenses can introduce geometric distortions that lead to an overestimate of the sample size by 3-10% To correct such aberrations, a system of lenses would be required, but even if such lenses could be found commercially, the range of available focal lengths, usable wavelengths and quality measures is limited. This leaves end-users with the expensive and time-consuming process of buying and trying
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