Integration of high-resolution imaging through scattering medium into a disposable micro-endoscope via projection of 2D spots-array.

The objective of this research includes integration of high-resolution imaging through scattering medium, such as blood, into a disposable micro-endoscope. A fiber laser integrated into the micro-endoscope as part of its illumination channel, allows to project a tunable array of spots of light onto an object, that is located behind the scattering medium. We have a laser fiber as part of the illumination channel of a disposable micro-endoscope. By using proper optics, we convert the temporal modulation of the laser into spatial distribution. Thus, the result is generation of spatial spots when using a pulsed laser. The detection channel is a holographic recording of the collected back scattered light, that allows extraction of the electrical field. By time integrating the field we obtain the realization of the spatial array of illumination spots formed on top of the inspected object and behind the scattering medium. By changing the temporal modulation of the illumination laser (changing its temporal photonic signals), we can tune the positions of the spots in the illumination array. If the distance between the projected spots is larger than the imaging resolution, then by applying localization microscopy algorithms together with scanning of the position of the spots in the array, will yield a high-resolution reconstruction of the inspected object. We theoretically and experimentally demonstrate the discussed operation principle and show the potential of the proposed concept as a modality in medical endoscopic procedures.

S194-Imaging through scattering media by 3D spatial filtering embedded into micro-endoscope.

BACKGROUND: The main objective is related to the capability of integrating into minimally invasive and ultra-thin disposable micro-endoscopic tool, a modality of realizing high-resolution imaging through scattering medium such as blood while performing medical procedure. In this research we aim for the first time to present a time-multiplexing super-resolving approach exhibiting enhanced focus sensitivity, generated by 3D spatial filtering, for significant contrast increase in images collected through scattering medium. METHOD: Our innovative method of imaging through scattering media provides imaging of only one specific object plane in scattering medium's volume while suppressing the noise coming from all other planes. The method should be assisted with axial scanning to perform imaging of the entire 3D object's volume. In our developed optical system noise suppression is achieved by 3D spatial filtering approach while more than an order of magnitude of suppression is experimentally demonstrated. The sensitivity to defocus and noise suppression is dramatically enhanced by placing an array of micro-lenses combined with pinholes raster positioned between two modules of telecentric lenses. RESULTS: We present our novel conceptual designs for the enhanced signal-to-noise ratio (SNR) when imaging through scattering medium and present preliminary experimental results demonstrating both quality imaging performed on resolution bars target as well as SNR quantified results in which SNR enhancement of more than one order of magnitude was obtained. CONCLUSIONS: In this paper, to the best of our knowledge, we present the first ever design of time-multiplexing-based approach for super-resolved imaging through scattering medium. The approach includes a time-multiplexing optical design significantly increasing the depth of focus sensitivity and after performing axial scanning yielding a significant enhancement of the SNR of the 3D object that is being imaged through the scattering medium. Right after the contrast (the SNR) enhancement we scan the object with the projected array of spots (raster) and map it continuously and with high imaging resolution.

Imaging of nanoparticle dynamics in live and apoptotic cells using temporally-modulated polarization.

Gold nanoparticles are widely exploited in phototherapy. Owing to their biocompatibility and their strong visible-light surface plasmonic resonance, these particles also serve as contrast agents for cell image enhancement and super-resolved imaging. Yet, their optical signal is still insufficiently strong for many important real-life applications. Also, the differentiation between adjacent nanoparticles is usually limited by the optical resolution and the orientations of non-spherical particles are unknown. These limitations hamper the progress in cell research by direct optical microscopy and narrow the range of phototherapy applications. Here we demonstrate exploiting the optical anisotropy of non-spherical nanoparticles to achieve super-resolution in live cell imaging and to resolve the intracellular nanoparticle orientations. In particular, by modulating the light polarization and taking advantage of the polarization-dependence of gold nanorod optical properties, we realize the 'lock-in amplification', widely-used in electronic engineering, to achieve image enhancement in live cells and in cells that undergo apoptotic changes.

Interference based localization of single emitters.

The ability to localize precisely a single optical emitter is important for particle tracking applications and super resolution microscopy. It is known that for a traditional microscope the ability to localize such an emitter is limited by the photon count. Here we analyze the ability to improve such localization by imposing interference fringes. We show here that a simple grating interferometer can introduce such improvement in certain circumstances and analyze what is required to increase the localization precision further.

Phase retrieval deblurring for imaging of dense object within a low scattering soft biological tissue.

Tissues are characterized by a strong scattering of visible optical radiation, which prevents one from achieving deep-tissue imaging. We propose a computational imaging technique for the inference of specific macroscopic, spatial phase distribution features of the scattering media. The spatial phase distribution is reconstructed from several defocused intensity images. We empirically demonstrate the method by reconstructing the location of two fibula chicken bones, embedded within chicken breast tissue. The suggested technique is safe, using visible laser illumination, and noninvasive. It is also cost-effective since a simple optical system is used and the images are acquired using a conventional camera, and it does not require interferometric detection as well as direct access to the object in absence of the layer.

K-factor image deshadowing for three-dimensional fluorescence microscopy.

The ability to track single fluorescent particles within a three dimensional (3D) cellular environment can provide valuable insights into cellular processes. In this paper, we present a modified nonlinear image decomposition technique called K-factor that reshapes the 3D point spread function (PSF) of an XYZ image stack into a narrow Gaussian profile. The method increases localization accuracy by ~60% with compare to regular Gaussian fitting, and improves minimal resolvable distance between overlapping PSFs by ~50%. The algorithm was tested both on simulated data and experimentally.

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.

Increased localization precision by interference fringe analysis.

We report a novel optical single-emitter-localization methodology that uses the phase induced by path length differences in a Mach-Zehnder interferometer to improve localization precision. Using information theory, we demonstrate that the localization capability of a modified Fourier domain signal generated by photon interference enables a more precise localization compared to a standard Gaussian intensity distribution of the corresponding point-spread function. The calculations were verified by numerical simulations and an exemplary experiment, where the centers of metal nanoparticles were localized to a precision of 3 nm.

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.

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).

Fast wide-field photothermal and quantitative phase cell imaging with optical lock-in detection.

We present a fast, wide-field holography system for detecting photothermally excited gold nanospheres with combined quantitative phase imaging. An interferometric photothermal optical lock-in approach (POLI) is shown to improve SNR for detecting nanoparticles (NPs) on multiple substrates, including a monolayer of NPs on a silanized coverslip, and NPs bound to live cells. Furthermore, the set up allowed for co-registered quantitative phase imaging (QPI) to be acquired in an off-axis holographic set-up. An SNR of 103 was obtained for NP-tagging of epidermal growth factor receptor (EGFR) in live cells with a 3 second acquisition, while an SNR of 47 was seen for 20 ms acquisition. An analysis of improvements in SNR due to averaging multiple frames is presented, which suggest that residual photothermal signal can be a limiting factor. The combination of techniques allows for high resolution imaging of cell structure via QPI with the ability to identify receptor expression via POLI.

Comparative review of interferometric detection of plasmonic nanoparticles.

Noble metal nanoparticles exhibit enhanced scattering and absorption at specific wavelengths due to a localized surface plamson resonance. This unique property can be exploited to enable the use of plasmonic nanoparticles as contrast agents in optical imaging. A range of optical techniques have been developed to detect nanoparticles in order to implement imaging schemes. Here we review several different approaches for using optical interferometry to detect the presence and concentration of nanoparticles. The strengths and weaknesses of the various approaches are discussed and quantitative comparisons of the achievable signal to noise ratios are presented. The benefits of each approach are outlined as they relate to specific application goals.

Photonic XOR with inherent loss compensation mechanism for memory cell implementation in a standard nanoscale very large-scale integrated fabrication process.

A multilayer photonic XOR gate is presented. The XOR is implemented by the interconnect layers of a microelectronic chip and is suitable for fabrication in a standard VLSI fabrication process. The proposed device features an inherent insertion loss compensation mechanism by utilization of nanometric holes, making it possible to implement an optic memory cell without the need of additional complex compensation devices. The structure of such a memory cell, implemented by utilization of two proposed XOR gates, configured to perform the NOT function, is shown. The unique structure of the proposed device allows us to significantly reduce sensitivity to process variations and therefore makes it possible to utilize the memory cell in state-of-the-art nanoscale processes. The proposed memory can be integrated with conventional electronics on the same VLSI chip.

Intercoupling surface plasmon resonance and diffusion reflection measurements for real-time cancer detection.

Spatial diffusion reflection (DR) measurements of gold nanorods (GNR) were recently suggested as a simple and highly sensitive non-invasive and non-ionizing method for real-time cancer detection. In this paper we demonstrate that wavelength dependent DR measurements enable the spectral red-shift observation of highly concentrated GNR. By conjugating targeting moieties to the GNR, large density of GNR can specifically home onto cancer cells. The inter-particle plasmon resonance pattern of the highly concentrated GNR leads to an extension and a red-shift (Δλ) in the absorption spectrum of the concentrated GNR. Dark-field microscopy was used in order to measure the expected Δλ in different GNR concentrations in vitro. Double-wavelength DR measurements of tissue-like phantoms and tumor bearing mice containing different GNR concentrations are presented. We show that the DR profile of the highly concentrated GNR directly correlate with the spectral extension and red-shift. This presented work suggests that wavelength dependent DR method can serve as a promising tool for real-time superficial tumor detection.

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.

Sub-micron particle based structures as reconfigurable photonic devices controllable by external photonic and magnetic fields.

In this paper we present the configurations of two nanometer scale structures--one of them optically controllable and the second one magnetically controllable. The first involves an array of nanoparticles that are made up of two layers (i.e., Au on top of a Si layer). The device may exhibits a wide range of plasmonic resonance according to external photonic radiation. The second type of device involves the usage of sub micron superparamagnetic particles located on a suitable structuring grid, that according to the angle of the external magnetic field allows control of the length of the structuring grid and therefore control the diffraction order of each wavelength.