Nir A. Turko

Wide-field interferometric phase microscopy with molecular specificity using plasmonic nanoparticles.

Abstract. We present a method for adding molecular specificity to wide-field interferometric phase microscopy (IPM) by recording the phase signatures of gold nanoparticles (AuNPs) labeling targets of interest in biological cells. The AuNPs are excited by time-modulated light at a wavelength corresponding to their absorption spectral peak, evoking a photothermal (PT) effect due to their plasmonic resonance. This effect induces a local temperature rise, resulting in local refractive index and phase changes that can be detected optically. Using a wide-field interferometric phase microscope, we acquired an image sequence of the AuNP sample phase profile without requiring lateral scanning, and analyzed the time-dependent profile of the entire field of view using a Fourier analysis, creating a map of the locations of AuNPs in the sample. The system can image a wide-field PT phase signal from a cluster containing down to 16 isolated AuNPs. AuNPs are then conjugated to epidermal growth factor receptor (EGFR) antibodies and inserted to an EGFR-overexpressing cancer cell culture, which is imaged using IPM and verified by confocal microscopy. To the best of our knowledge, this is the first time wide-field interferometric PT imaging is performed at the subcellular level without the need for total internal reflection effects or scanning.

Flipping interferometry and its application for quantitative phase microscopy in a micro-channel.

We present a portable, off-axis interferometric module for quantitative phase microscopy of live cells, positioned at the exit port of a coherently illuminated inverted microscope. The module creates on the digital camera an interference pattern between the image of the sample and its flipped version. The proposed simplified module is based on a retro-reflector modification in an external Michelson interferometer. The module does not contain any lenses, pinholes, or gratings and its alignment is straightforward. Still, it allows full control of the off-axis angle and does not suffer from ghost images. As experimentally demonstrated, the module is useful for quantitative phase microscopy of live cells rapidly flowing in a micro-channel.

Linear and nonlinear optical waveguiding in bio-inspired peptide nanotubes.

Unique linear and nonlinear optical properties of bioinspired peptide nanostructures such as wideband transparency and high second-order nonlinear optical response, combined with elongated tubular shape of variable size and rapid self-assembly fabrication process, make them promising for diverse bio-nano-photonic applications. This new generation of nanomaterials of biological origin possess physical properties similar to those of biological structures. Here, we focus on new specific functionality of ultrashort peptide nanotubes to guide light at fundamental and second-harmonic generation (SHG) frequency in horizontal and vertical peptide nanotubes configurations. Conducted simulations and experimental data show that these self-assembled linear and nonlinear optical bio-waveguides provide strong optical power confinement factor, demonstrate pronounced directionality of SHG and high conversion efficiency of SHG ∼10(-5). Our study gives new insight on physics of light propagation in nanostructures of biological origin and opens the avenue towards new and unexpected applications of these waveguiding effects in bio-nanomaterials both for biomedical nonlinear microscopy imaging recognition and development of novel integrated nanophotonic devices.

Simultaneous two-wavelength phase unwrapping using an external module for multiplexing off-axis holography

We present a dual-wavelength external holographic microscopy module for quantitative phase imaging of 3D structures with extended thickness range. This is done by simultaneous acquisition of two off-axis interferograms, each at a different wavelength, and generation of a synthetic wavelength, which is larger than the sample optical thickness, allowing two-wavelength unwrapping. The simultaneous acquisition is carried out by using optical multiplexing of the two interferograms onto the camera, where each of them has orthogonal off-axis interference fringe direction in relation to the other one. We used the system to quantitatively image a 7.96 μm step target and 30.5 μm circular copper pillars.

Optimal spatial bandwidth capacity in multiplexed off-axis holography for rapid quantitative phase reconstruction and visualization

We present new methods for optimizing the spatial bandwidth capacity in off-axis holography using spatial multiplexing. We use optimal spatial multiplexing of off-axis holograms to fill the entire spatial frequency domain, including the space previously occupied by the intensity of the sample. Our approach enables spatial digital compression of eight off-axis holograms into a single real-valued multiplexed hologram, having the same number of pixels as each of the input holograms, but still allowing their full reconstruction without resolution or magnification loss in the reconstructed complex wave fronts. This new method allows 33% improvement in usage of the spatial bandwidth capacity compared to the best available off-axis holography real-value multiplexing method. Since the output multiplexed hologram contains only real values, it can be used for rapid display of eight wave front reconstructions at once, which is useful for real-time visualization, when the hologram display device is slower than the acquiring camera. We further generalize this technique to digital multiplexing of 16 real-valued holograms into a single complex-valued hologram by simple arithmetic operations in the hologram domain. Then, the extraction of the 16 wave fronts includes a single 2-D discrete Fourier transform to access the spatial frequency domain, allowing fast reconstruction, which is useful for real-time processing of off-axis holograms, with improved processing rate compared to current hologram processing algorithms. These new approaches allow the full reconstruction of all compressed data without loss of resolution or magnification, even though the samples are dense such that their frequency content employs the entire range. Both multiplexing architectures are then demonstrated for experimentally-acquired off-axis holograms for quantitative phase imaging of biological cells.

Dynamic measurements of flowing cells labeled by gold nanoparticles using full-field photothermal interferometric imaging

Abstract. We present highly dynamic photothermal interferometric phase microscopy for quantitative, selective contrast imaging of live cells during flow. Gold nanoparticles can be biofunctionalized to bind to specific cells, and stimulated for local temperature increase due to plasmon resonance, causing a rapid change of the optical phase. These phase changes can be recorded by interferometric phase microscopy and analyzed to form an image of the binding sites of the nanoparticles in the cells, gaining molecular specificity. Since the nanoparticle excitation frequency might overlap with the sample dynamics frequencies, photothermal phase imaging was performed on stationary or slowly dynamic samples. Furthermore, the computational analysis of the photothermal signals is time consuming. This makes photothermal imaging unsuitable for applications requiring dynamic imaging or real-time analysis, such as analyzing and sorting cells during fast flow. To overcome these drawbacks, we utilized an external interferometric module and developed new algorithms, based on discrete Fourier transform variants, enabling fast analysis of photothermal signals in highly dynamic live cells. Due to the self-interference module, the cells are imaged with and without excitation in video-rate, effectively increasing signal-to-noise ratio. Our approach holds potential for using photothermal cell imaging and depletion in flow cytometry.

Simultaneous three-wavelength unwrapping using external digital holographic multiplexing module

We present an external interferometric setup that is able to simultaneously acquire three wavelengths of the same sample instance without scanning or multiple exposures. This setup projects onto the monochrome digital camera three off-axis holograms with rotated fringe orientations, each from a different wavelength channel, without overlap in the spatial-frequency domain, and thus allows the full reconstruction of the three complex wavefronts from the three wavelength channels. We use this new setup for three-wavelength phase unwrapping, allowing phase imaging of thicker objects than possible with a single wavelength, but without the increased level of noise. We demonstrate the proposed technique for micro-channel profiling and label-free cell imaging.

Single-exposure full-field multi-depth imaging using low-coherence holographic multiplexing

We present a new interferometric imaging approach that allows for multiple-depth imaging in a single acquisition, using off-axis low-coherence holographic multiplexing. This technique enables sectioned imaging of multiple slices within a thick sample, in a single image acquisition. Each slice has a distinct off-axis interference fringe orientation indicative of its axial location, and the camera acquires the multiplexed hologram containing the different slices at once. We demonstrate the proposed technique for amplitude and phase imaging of optically thick samples.

Quantitative study of optical and mechanical bone status using multispectral photoacoustics

Osteoporosis is a major public health problem worldwide. Here, we present a quantitative multispectral photoacoustic method for the evaluation of bone pathologies which has significant advantages over pure ultrasonic or pure optical methods as it provides both molecular information and bone mechanical status. This is enabled via a simultaneous measurement of the bones optical properties as well as the speed of sound and ultrasonic attenuation in the bone. To test the methods quantitative predictions, a combined ultrasonic and photoacoustic system was developed. Excitation was performed optically via a portable triple laser-diode system and acoustically via a single element transducer. Additional dual transducers were used for detecting the acoustic waves that were generated by the two modalities. Both temporal and spectral parameters were compared between different excitation wavelengths and measurement modalities. Short photoacoustic excitation wavelengths allowed sensing of the cortical layer while longer wavelengths produced results which were compatible with the quantitative ultrasound measurements.

Photothermal quantitative phase imaging of living cells with nanoparticles utilizing a cost-efficient setup

We explored photothermal quantitative phase imaging (PTQPI) of living cells with functionalized nanoparticles (NPs) utilizing a cost-efficient setup based on a cell culture microscope. The excitation light was modulated by a mechanical chopper wheel with low frequencies. Quantitative phase imaging (QPI) was performed with Michelson interferometer-based off-axis digital holographic microscopy and a standard industrial camera. We present results from PTQPI observations on breast cancer cells that were incubated with functionalized gold NPs binding to the epidermal growth factor receptor. Moreover, QPI was used to quantify the impact of the NPs and the low frequency light excitation on cell morphology and viability.