24 February 2010 Practical optical quality assessment and correction of a nonlinear microscope
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Nonlinear microscopy (NLM) has covered the requirement for higher contrast and resolution compared with other microscopy techniques, however, the optical quality of this imaging apparatus and the sample structure can compromise its capabilities. Here, we show that the imaging capabilities of a NLM can be affected by the aberrations produced by the setup optical elements alignment, the materials from which they are fabricated and more importantly by the sample. To overcome this, a Shack-Hartman Wavefront sensing scheme has been implemented for characterizing: a) the whole NLM setup and, b) the sample induced aberrations. The first part includes all the aberrations introduced by the optical elements, starting from the laser and until the microscope objective. Having these information, aberrations can be compensated in a closed-loop configuration resulting in the system calibration. Then the remaining aberrations (microscope objective and sample) are recorded. This is done employing the sample nonlinear fluorescence signal collected at one point (keeping the excitation beam static) in the imaging plane. Given that this emission is an incoherent process, it can be considered as a point source. Therefore its wavefront will contain the sample and the objective aberrations. Using the wavefront sensor the information is recorded and passed to the deformable mirror which will compensate the aberrations in a "single shot" (open-loop configuration). This compared with other adaptive optics strategies (i.e. iterative algorithms) results in a reduced sample exposure, and greatly decreases sample damage. Importantly the application of both corrections (system and sample) enables a significant signal intensity and contrast improvement.
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Rodrigo Aviles-Espinosa, Rodrigo Aviles-Espinosa, Jordi Andilla, Jordi Andilla, Rafael Porcar-Guezenec, Rafael Porcar-Guezenec, Omar Olarte, Omar Olarte, Susana I.C. O. Santos, Susana I.C. O. Santos, Xavier Levecq, Xavier Levecq, David Artigas, David Artigas, Pablo Loza-Alvarez, Pablo Loza-Alvarez, } "Practical optical quality assessment and correction of a nonlinear microscope", Proc. SPIE 7570, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVII, 75700W (24 February 2010); doi: 10.1117/12.840978; https://doi.org/10.1117/12.840978

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