Some biological experiments demand the observation of dynamics processes in 3D with high spatiotemporal resolution. The use of wavefront coding to extend the depth-of-field (DOF) of the collection arm of a light-sheet microscope is an interesting alternative for fast 3D imaging. Under this scheme, the 3D features of the sample are captured at high volumetric rates while the light sheet is swept rapidly within the extended DOF. The DOF is extended by coding the pupil function of the imaging lens by using a custom-designed phase mask. A posterior restoration step is required to decode the information of the captured images based on the applied phase mask . This hybrid optical-digital approach is known as wavefront coding (WFC). Previously, we have demonstrated this method for performing fast 3D imaging of biological samples at medium resolution . In this work, we present the extension of this approach for high-resolution microscopes. Under these conditions, the effective DOF of a standard high NA objective is of a few micrometers. Here we demonstrate that by the use of WFC, we can extend the DOF more than one order of magnitude keeping the high-resolution imaging. This is demonstrated for two designed phase masks using Zebrafish and C. elegans samples.
 Olarte, O.E., Andilla, J., Artigas, D., and Loza-Alvarez, P., “Decoupled Illumination-Detection Microscopy. Selected Optics in Year 2105,” in Optics and Photonics news 26, p. 41 (2015).
 Olarte, O.E., Andilla, J., Artigas, D., and Loza-Alvarez, P., “Decoupled illumination detection in light sheet microscopy for fast volumetric imaging,” Optica 2(8), 702 (2015).
Acquisition of images deep inside large samples is one of the most demanded improvements that current biology
applications ask for. Absorption, scattering and optical aberrations are the main difficulties encountered in these types of
samples. Adaptive optics has been imported form astronomy to deal with the optical aberrations induced by the sample.
Nonlinear microscopy and SPIM have been proposed as interesting options to image deep into a sample. Particularly,
light-sheet microscopy, due to its low photo bleaching properties, opens new opportunities to obtain information for
example in long time lapses for large 3D imaging. In this work, we perform an overview of the application of adaptive
optics to the fluorescence microscopy in linear and non-linear modalities. Then we will focus in the light-sheet
microscopy architecture of two orthogonal optical paths which implies new requirements in terms of optical correction.
We will see the different issues that appear in light-sheet microscopy particularly when imaging large and non-flat
samples. Finally, we will study the problem of the isoplanetic patches.
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