Anisoplanatic adaptive optics in parallelized laser scanning microscopy.

Inhomogeneities in the refractive index of a biological microscopy sample can introduce phase aberrations, severely impairing the quality of images. Adaptive optics can be employed to correct for phase aberrations and improve image quality. However, conventional adaptive optics can only correct a single phase aberration for the whole field of view (isoplanatic correction) while, due to the highly heterogeneous nature of biological tissues, the sample induced aberrations in microscopy often vary throughout the field of view (anisoplanatic aberration), limiting significantly the effectiveness of adaptive optics. This paper reports on a new approach for aberration correction in laser scanning confocal microscopy, in which a spatial light modulator is used to generate multiple excitation points in the sample to simultaneously scan different portions of the field of view with completely independent correction, achieving anisoplanatic compensation of sample induced aberrations, in a significantly shorter time compared to sequential isoplanatic correction of multiple image subregions. The method was tested in whole Drosophila brains and in larval Zebrafish, each showing a dramatic improvement in resolution and sharpness when compared to conventional isoplanatic adaptive optics.

Pupil mask diversity for image correction in microscopy.

Three-dimensional microscopy suffers from sample-induced aberrations that reduce the resolution and lead to misinterpretations of the object distribution. In this paper, the resolution of a three-dimensional fluorescent microscope is significantly improved by introducing an amplitude diversity in the form of a binary amplitude mask positioned in several different orientations within the pupil, followed by computer processing of the diversity images. The method has proved to be fast, easy to implement, and cost-effective in high-resolution imaging of casper fli:GFP zebrafish.

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.

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.

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.

Dressing plasmons in particle-in-cavity architectures.

Placing metallic nanoparticles inside cavities, rather than in dimers, greatly improves their plasmonic response. Such particle-in-cavity (PIC) hybrid architectures are shown to produce extremely strong field enhancement at the particle-cavity junctions, arising from the cascaded focusing of large optical cross sections into small gaps. These simply constructed PIC structures produce the strongest field enhancement for coupled nanoparticles, up to 90% stronger than for a dimer. The coupling is found to follow a universal power law with particle-surface separation, both for field enhancements and resonant wavelength shifts. Significantly enhanced Raman signals are experimentally observed for molecules adsorbed in such PIC structures, in quantitive agreement with theoretical calculations. PIC architectures may have important implications in many applications, such as reliable single molecule sensing and light harvesting in plasmonic photovoltaic devices.

High-speed 2D and 3D fluorescence microscopy of cardiac myocytes.

Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture microscope objective to both illuminate a tilted plane within the specimen and to obtain an image of the tilted illuminated plane. In this paper, we present a new OPM configuration that enables both the illumination and detection focal planes to be swept simultaneously and remotely through the sample volume, enabling high speed volumetric imaging. We demonstrate the high speed imaging capabilities of the system by imaging calcium dynamics in cardiac myocytes in 2D at 926 frames per second and in 3D at 21 volumes per second. In the future, higher frame rate CCD cameras will enable volumetric imaging at much greater rates, leading to new capabilities to study dynamic events in cells at high speeds in two and three dimensions.

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.