Paolo Pozzi

Pupil filters for extending the field-of-view in light-sheet microscopy.

Pupil filters, represented by binary phase modulation, have been applied to extend the field of view of a light-sheet fluorescence microscope. Optimization has been used, first numerically to calculate the optimum filter structure and then experimentally, to scale and align the numerically synthesized filter in the microscope. A significant practical extension of the field of view has been observed, making the reported approach a valuable tool on the path to wide-field light-sheet microscopy.

Adaptive illumination based on direct wavefront sensing in a light-sheet fluorescence microscope

A methodology for the adaptive control and correction of phase aberrations in the illumination arm of a light-sheet fluorescence microscope has been developed. The method uses direct wavefront sensing on epi-fluorescent light to detect the aberration present in the sample. Using this signal, the aberrations in the illumination arm are subsequently corrected with a spatial light modulator in a feedforward mode. Adaptive correction, resulting in significant improvement in the axial resolution, has been demonstrated by imaging Tg(fli:GFP) zebrafish embryos.

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

Lensless coherent imaging by sampling of the optical field with digital micromirror device

We have experimentally demonstrated a lensless coherent microscope based on direct registration of the complex optical field by sampling the pupil with a sequence of two-point interferometers formed by a digital micromirror device. Complete registration of the complex amplitude in the pupil of the imaging system, without any reference beam, provides a convenient link between the experimental and computational optics. Unlike other approaches to digital holography, our method does not require any external reference beam, resulting in a simple and robust registration setup. Computer analysis of the experimentally registered field allows for focusing the image in the whole range from zero to infinity, and for virtual correction of the aberrations present in the real optical system, by applying the adaptive wavefront corrections to its virtual model.

Holographic imaging with a Shack-Hartmann wavefront sensor

A high-resolution Shack-Hartmann wavefront sensor has been used for coherent holographic imaging, by computer reconstruction and propagation of the complex field in a lensless imaging setup. The resolution of the images obtained with the experimental data is in a good agreement with the diffraction theory. Although a proper calibration with a reference beam improves the image quality, the method has a potential for reference-less holographic imaging with spatially coherent monochromatic and narrowband polychromatic sources in microscopy and imaging through turbulence.

Optical path difference microscopy with a Shack–Hartmann wavefront sensor

In this Letter, we show that a Shack-Hartmann wavefront sensor can be used for the quantitative measurement of the specimen optical path difference (OPD) in an ordinary incoherent optical microscope, if the spatial coherence of the illumination light in the plane of the specimen is larger than the microscope resolution. To satisfy this condition, the illumination numerical aperture should be smaller than the numerical aperture of the imaging lens. This principle has been successfully applied to build a high-resolution reference-free instrument for the characterization of the OPD of micro-optical components and microscopic biological samples.

Phase retrieval from multiple binary masks generated speckle patterns

We present a reference-less and time-multiplexing phase retrieval method by making use of the digital micromirror device (DMD). In this method, the DMD functions not only as a flexible binary mask which modulates the optical field, but also as a sampling mask for measuring corresponding phases, which makes the whole setup simple and robust. The DMD reflection forms a sparse intensity mask in the pupil which produces speckle pattern after propagation. With the recorded intensity on the camera and the binary pattern on the DMD, the phase in all the ‘on’ pixels can be reconstructed at once by solving inverse problems with iterative methods, for instance using Gerchberg-Saxton algorithm. Then the phase of the whole pupil can be reconstructed from a series of binary patterns and speckle patterns. Numerical experiments show the feasibility of this phase retrieval method and the importance of sparse binary masks in the improving of convergence speed.

Feedforward operation of a lens setup for large defocus and astigmatism correction

In this manuscript, we present a lens setup for large defocus and astigmatism correction. A deformable defocus lens and two rotational cylindrical lenses are used to control the defocus and astigmatism. The setup is calibrated using a simple model that allows the calculation of the lens inputs so that a desired defocus and astigmatism are actuated on the eye. The setup is tested by determining the feedforward prediction error, imaging a resolution target, and removing introduced aberrations.

Optimal model-based sensorless adaptive optics for epifluorescence microscopy

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.