Oscar Azucena

Adaptive optics confocal microscopy using direct wavefront sensing

Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images in deep-tissue imaging. We introduce a confocal fluorescence microscope with adaptive optics, which can correct aberrations based on direct wavefront measurements using a Shack-Hartmann wavefront sensor with a fluorescent bead used as a point source reference beacon. The results show a 4.3× improvement in the Strehl ratio and a 240% improvement in the signal intensity for fixed mouse tissues at depths of up to 100 μm.

Adaptive optics wide-field microscopy using direct wavefront sensing

We report a technique for measuring and correcting the wavefront aberrations introduced by a biological sample using a Shack-Hartmann wavefront sensor, a fluorescent reference source, and a deformable mirror. The reference source and sample fluorescence are at different wavelengths to separate wavefront measurement and sample imaging. The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the structure of the sample to be resolved.

Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons

We present a new method to directly measure and correct the aberrations introduced when imaging through thick biological tissue. A Shack-Hartmann wavefront sensor is used to directly measure the wavefront error induced by a Drosophila embryo. The wavefront measurements are taken by seeding the embryo with fluorescent microspheres used as “artificial guide-stars.” The wavefront error is corrected in ten millisecond steps by applying the inverse to the wavefront error on a micro-electro-mechanical deformable mirror in the image path of the microscope. The results show that this new approach is capable of improving the Strehl ratio by 2 times on average and as high as 10 times when imaging through 100 μm of tissue. The results also show that the isoplanatic half-width is approximately 19 μm resulting in a corrected field of view 38 μm in diameter around the guide-star.

Live imaging using adaptive optics with fluorescent protein guide-stars.

Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy.

Adaptive optics microscopy with direct wavefront sensing using fluorescent protein guide stars

We introduce a direct wavefront sensing method using structures labeled with fluorescent proteins in tissues as guide stars. An adaptive optics confocal microscope using this method is demonstrated for imaging of mouse brain tissue. A dendrite and a cell body of a neuron labeled with yellow fluorescent protein are tested as guide stars without injection of other fluorescent labels. Photobleaching effects are also analyzed. The results shows increased image contrast and 3× improvement in the signal intensity for fixed mouse tissues at depths of 70 μm.

Inkjet printing of passive microwave circuitry

Inkjet printing technology was utilized to fabricate transmission lines on a glass substrate. 50 micron resolution was realized using 10 pL drop volumes on a Corning 7740 glass substrate. This can be further improved by applying other methods as described in this paper. The conductivity of the sintered silver structures were 1/6 that of bulk silver after sintering at a temperature much lower than the melting point of bulk silver. A comparison of the DC resistance of the sintered silver shows that it can be a match for electroplated and etched copper. Printed Coplanar lines demonstrated losses of 1.62 dB/cm at 10 GHZ and 2.65 dB/cm at 20 GHz.

Adaptive optical two-photon microscopy using autofluorescent guide stars

We demonstrate a fast, direct wavefront-sensing method for dynamic in vivo adaptive optical two-photon microscopy. By using a Shack-Hartmann wavefront sensor and open-loop control, the system provides high-speed wavefront measurement and correction. To measure the wavefront in the middle of a Drosophila embryo at early stages, autofluorescence from endogenous fluorophores in the yolk were used as reference guide stars. The method was tested through live imaging of a Drosophila embryo. The aberration in the middle of the embryo was measured directly for the first time. After correction, the contrast and signal intensity of the structure in the middle of the embryo was improved.

Shack-Hartmann wavefront sensing using interferometric focusing of light onto guide-stars

Optical microscopy provides noninvasive imaging of biological tissues at subcellular level. The optical aberrations induced by the inhomogeneous refractive index of biological samples limits the resolution and can decrease the penetration depth. To compensate refractive aberrations, adaptive optics with Shack-Hartmann wavefront sensing has been used in microscopes. Wavefront measurement requires light from a guide-star inside of the sample. The scattering effect limits the intensity of the guide-star, hence reducing the signal to noise ratio of the wavefront measurement. In this paper, we demonstrate the use of interferometric focusing of excitation light onto a guide-star embedded deeply in tissue to increase its fluorescent intensity, thus overcoming the excitation signal loss caused by scattering. With interferometric focusing, we more than doubled the signal to noise ratio of the laser guide-star through scattering tissue as well as potentially extend the imaging depth through using AO microscopy.

The design and optimization of detectors for adaptive optics wavefront sensing

The most common detector configuration for Shack Hartmann (SH) wavefront sensors used for adaptive optics (AO) wavefront sensing is the quad cell. Advances in detectors, such as the CCDs being developed in a project on which we are collaborators (funded by the Adaptive Optics Development Program), make it possible to use larger pixel arrays. The CCD designs incorporate improved read amplifiers and novel pixel geometries optimized for laser guide star (LGS) AO wavefront sensing. While it is likely that finer sampling of the SH spot will improve the ability of the wavefront sensor to accurately determine the spot displacement, particularly for elongated or aberrated spots such as those seen in LGS AO systems, the optimal sampling is not dependent simply on the number of pixels but must also take into account the effects of photon and detector noise. The performance of a SH wavefront sensor also depends on the performance of the algorithm used to find the spot displacement. In the literature alternatives have been proposed to the common center of mass algorithm, but these have not been simulated in detail. In this paper we will describe the results of our study of the performance of a SH wavefront sensor with a well sampled spot. We will present results for simulations of the wavefront sensor that enable us to optimize the design of the detector for varying conditions of signal to noise and spot elongation. We will also discuss the application of correlation algorithms to SH wavefront sensors and present results regarding the performance and statistics of this algorithm.

Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction

Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon [1]. AO has also been applied to vision science to improve the view of the human eye. This paper will address our current research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology. A Shack-Hartmann wavefront sensor (SHWS) is used to measure the aberration introduced by a Drosophila Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon. Previous measurements of the wavefront aberrations have found an average peak-to-valley and root-mean-square (RMS) wavefront error of 0.77 micrometers and 0.15 micrometers, respectively. Measurements of the Zernike coefficients indicated that the correction of the first 14 Zernike coefficients is sufficient to correct the aberrations we measured. Here we show that a MEMS deformable mirror with 3.5 microns of stroke and 140 actuators is sufficient to correct these aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted fluorescent reference beacon for wavefront correction are discussed.