Joel Kubby

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.

High-speed scanning interferometric focusing by fast measurement of binary transmission matrix for channel demixing

Using the fast measurement of a binary transmission matrix and a digital micromirror device, we demonstrate high-speed interferometric focusing through highly dynamic scattering media with binary intensity modulation. The scanning of speckles for reference optimization gives stable focusing, which can be used for focusing through a fast changing media or two dimensional scanning through a slowly changing scattering media. The system allows dynamic focusing at 12.5 Hz with 1024 input modes, and more than 60 times intensity enhancement. It was tested with a moving diffuser, a mouse brain and skull tissue. The experiment with a live drosophila embryo shows its potential in compensating dynamic scattering in live biological tissue.

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.

Transcutical imaging with cellular and subcellular resolution

We demonstrate transcutical structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins and calcium indicators in the living Drosophila brain with cellular and subcellular resolution.