Tali Ilovitsh

Cellular imaging using temporally flickering nanoparticles

Utilizing the surface plasmon resonance effect in gold nanoparticles enables their use as contrast agents in a variety of applications for compound cellular imaging. However, most techniques suffer from poor signal to noise ratio (SNR) statistics due to high shot noise that is associated with low photon count in addition to high background noise. We demonstrate an effective way to improve the SNR, in particular when the inspected signal is indistinguishable in the given noisy environment. We excite the temporal flickering of the scattered light from gold nanoparticle that labels a biological sample. By preforming temporal spectral analysis of the received spatial image and by inspecting the proper spectral component corresponding to the modulation frequency, we separate the signal from the wide spread spectral noise (lock-in amplification).

Self-assembled monolayer assisted bonding of Si and InP

A versatile procedure for the low-temperature bonding of silicon and indium-phosphide to silicon is proposed and demonstrated. The procedure relies on the deposition and functionalization of self-assembled, single molecular layers on the surface of one substrate, and the subsequent attachment of the monolayer to the surface of the other substrate with or without its own monolayer coating. The process is applicable to the fabrication of hybrid-silicon, active photonic devices.

Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing

Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.

Cellular superresolved imaging of multiple markers using temporally flickering nanoparticles

In this paper we present a technique aimed for simultaneous detection of multiple types of gold nanoparticles (GNPs) within a biological sample, using lock-in detection. We image the sample using a number of modulated laser beams that correspond to the number of GNP species that label a given sample. The final image where the GNPs are spatially separated is obtained computationally. The proposed method enables the simultaneous superresolved imaging of different areas of interest within biological sample and also the spatial separation of GNPs at sub-diffraction distances, making it a useful tool in the study of intracellular trafficking pathways in living cells.

Superresolved labeling nanoscopy based on temporally flickering nanoparticles and the K-factor image deshadowing

Localization microscopy provides valuable insights into cellular structures and is a rapidly developing field. The precision is mainly limited by additive noise and the requirement for single molecule imaging that dictates a low density of activated emitters in the field of view. In this paper we present a technique aimed for noise reduction and improved localization accuracy. The method has two steps; the first is the imaging of gold nanoparticles that labels targets of interest inside biological cells using a lock-in technique that enables the separation of the signal from the wide spread spectral noise. The second step is the application of the K-factor nonlinear image decomposition algorithm on the obtained image, which improves the localization accuracy that can reach 5nm and enables the localization of overlapping particles at minimal distances that are closer by 65% than conventional methods.

Super-resolution using Barker-based array projected via spatial light modulator

The use of a two-dimensional Barker-based array in the conventional time multiplexing super-resolution (TMSR) technique was recently presented [Opt. Lett.40, 163-165 (2015)OPLEDP0146-959210.1364/OL.40.000163]. It enables achieving a two-dimensional SR image using only a one-dimensional scan, by exploiting its unique auto-correlation property. In this Letter, we refine the method using a mismatched array for the decoding process. The cross-correlation between the Barker-based array and the mismatched array has a perfect peak-to-sidelobes ratio, making it ideal for the SR process. Also, we propose the projection of this array onto the object using a phase-only spatial light modulator. Projecting the array eliminates the need for printing it, mechanically shifting it, and having a direct contact with the object, which is not feasible in many imaging applications. 13 phase masks, which generate shifted Barker-based arrays, were designed using a revised Gerchberg-Saxton algorithm. A sequence of 13 low resolution images were captured using these phase masks, and were decoded using the mismatched arrays, resulting in a high-resolution image. The proposed mismatched array and the design process of the phase masks are presented, and the method is validated by a laboratory experiment.

Superresolved nanoscopy using Brownian motion of fluorescently labeled gold nanoparticles

The fundamental limit set by the wavelength of light can be overcome using methods of superresolution localization microscopy. These methods require labeling of the sample with fluorescent molecules and are time consuming as repeated cycles of activation and photobleaching of the sample are required. Alternatively, we propose a simplified approach that is free from direct labeling with fluorescence molecules and does not require the repeated cycles of activation and photobleaching. The method uses fluorescently labeled gold nanoparticles in an aqueous solution that are distributed on top of the sample. The nanoparticles move in random Brownian motion and obscure different areas of the sample, while the scene is being imaged sequentially. By conducting the proper postprocessing, a superresolution image can be generated. The method is validated both by numerical simulations as well as by experimental data.

Silicon-coated gold nanoparticles nanoscopy

Abstract. This paper presents a method for modifying the point spread function (PSF) into a doughnut-like shape, through the utilization of the plasma dispersion effect (PDE) of silicon-coated gold nanoparticles. This modified PSF has spatial components smaller than the diffraction limit, and by scanning the sample with it, super-resolution can be achieved. The sample is illuminated using two laser beams. The first is the pump, with a wavelength in the visible region that creates a change in the refractive index of the silicon coating due to the PDE. This creates a change in the localized surface plasmon resonance wavelength. Since the pump beam has a Gaussian profile, the high intensity areas of the beam experience the highest refractive index change. When the second beam (i.e., the probe) illuminates the sample with a near-infrared wavelength, this change in the refractive index is transformed into a change in the PSF profile. The ordinary Gaussian shape is transformed into a doughnut shape, with higher spatial frequencies, which enables one to achieve super-resolution by scanning the specimen using this PSF. This is a step toward the creation of a nonfluorescent nanoscope.

Phase stretch transform for super-resolution localization microscopy

Super-resolution localization microscopy has revolutionized the observation of living structures at the cellular scale, by achieving a spatial resolution that is improved by more than an order of magnitude compared to the diffraction limit. These methods localize single events from isolated sources in repeated cycles in order to achieve super-resolution. The requirement for sparse distribution of simultaneously activated sources in the field of view dictates the acquisition of thousands of frames in order to construct the full super-resolution image. As a result, these methods have slow temporal resolution which is a major limitation when investigating live-cell dynamics. In this paper we present the use of a phase stretch transform for high-density super-resolution localization microscopy. This is a nonlinear frequency dependent transform that emulates the propagation of light through a physical medium with a specific warped diffractive property and applies a 2D phase function to the image in the frequency domain. By choosing properly the transform parameters and the phase kernel profile, the point spread function of each emitter can be sharpened and narrowed. This enables the localization of overlapping emitters, thus allowing a higher density of activated emitters as well as shorter data collection acquisition rates. The method is validated by numerical simulations and by experimental data obtained using a microtubule sample.

Optical realization of the radon transform

This paper presents a novel optical system for the realization of the Radon transform in a single frame. The optical system is simple, fast and accurate and consists of a 4F system, where in the 2F plane a vortex like optical element is placed. This optical element performs the rotation of the object, which replaces the need for mechanically rotating it, as is done in other common optical realization techniques of the Radon transform. This optical element is realized using a spatial light modulator (SLM) and an amplitude slide. The obtained Radon transform is given in Cartesian coordinates, which can subsequently be transformed using a computer to a polar set. The proposed concept is supported mathematically, numerically and experimentally.