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Current serological diagnostic tests for Zika virus (ZIKV) suffer from reduced sensitivity, high cross-reactivity, low specificity, and lengthy protocols. Here, we begin by reviewing serological and antigenemia assays that are either commercially available or under development. We then focus on a new technology, named magnetic modulation biosensing (MMB), that enables rapid detection of ZIKV immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. The MMB system utilizes magnetic beads that are conjugated to recombinant ZIKV nonstructural 1 (NS1) protein, which specifically captures Zika IgM/IgG antibodies. A second fluorescently labeled antibody is then added, forming a “sandwich” with the analyte and the capture protein. An oscillating magnetic field gradient concentrates the beads within the sample volume and transports them in a periodic motion in and out of a laser beam, producing an oscillating signal that is detected and demodulated. Compared with EUROIMMUN enzyme-linked immunosorbent assay (ELISA), MMB exhibits a much higher clinical sensitivity (88%–97% vs 38%–74%), a much longer time after symptom onset in which the IgM antibodies can be detected (5–180 vs 7–29 days post symptom onset), and a lower false positive rate. Additionally the specificity is 100%, and the cross-reactivity with West Nile and dengue viruses is minimal (0%–4%). The sensitivity, specificity, and simplicity of the MMB assays can significantly improve Zika diagnosis and provide accurate results for public health agencies.

Background Zika virus has created global alarm because it has been associated with catastrophic fetal abnormalities, including microcephaly, spontaneous abortion, and intrauterine growth restriction. Current serological assays that detect antiviral antibodies suffer from low sensitivity and high cross-reactivity among different flaviviruses. Methods In this study, utilizing a novel magnetic modulation biosensing (MMB) system and the Zika nonstructural 1 protein, we show highly sensitive and specific Zika serological assays. We blindly tested 60 reverse-transcription polymerase chain reaction Zika-positive samples and healthy patients' serum samples, as well as 44 serum samples from enzyme-linked immunosorbent assay (ELISA) West Nile- and dengue-positive patients. The Zika-positive samples were collected from Israeli travelers returning from Zika-endemic areas. Results The MMB Zika assays have 88%-97% sensitivity, much higher than the current state-of-the-art EUROIMMUN ELISA assays (38%-74%). In addition, the specificity is 100%, and the cross-reactivity with West Nile and dengue viruses is minimal (0%-4%). Furthermore, the MMB assays detected Zika IgM antibodies as early as 5 days and as late as 180 days postsymptoms onset, significantly extending the number of days that the antibodies are detectable. Conclusions The sensitivity, specificity, and simplicity of the MMB assays may significantly improve Zika diagnosis and provide accurate results for public health agencies.

Rapid and precise manipulation of magnetic beads on the nano and micro scales is essential in many biosensing applications, such as separating target molecules from background molecules and detecting specific proteins and DNA sequences in plasma. Accurately moving magnetic beads back and forth requires at least two adjustable magnetic field gradients. Unlike permanent magnets, electromagnets are easy to design and can produce strong and adjustable magnetic field gradients without mechanical motion, making them desirable for use in robust and safe medical devices. However, using multiple magnetic field sources to manipulate magnetic beads presents several challenges, including overlapping magnetic fields, added bulk, increased cost, and reduced durability. Here, we provide a thorough analysis, including analytical calculations, numerical simulations, and experimental measurements, of using two electromagnets to manipulate magnetic beads inside a miniature glass cell. We analyze and experimentally demonstrate different aspects of the electromagnets&rsquo, design, such as their mutual influence, the advantages and disadvantages of different pole tip geometries, and the correlation between the electromagnets&rsquo, positions and the beads&rsquo, aggregation during movement. Finally, we have devised a protocol to maximize the magnetic forces acting on magnetic beads in a two-electromagnet setup while minimizing the electromagnets&rsquo, size. We used two such electromagnets in a small footprint magnetic modulation biosensing system and detected as little as 13 ng/L of recombinant Zika virus antibodies, which enables detection of Zika IgM antibodies as early as 5 days and as late as 180 days post symptoms onset, significantly extending the number of days that the antibodies are detectable.

Intravital microscopy techniques have become increasingly important in biomedical research because they can provide unique microscopic views of various biological or disease developmental processes in situ. Here we present an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system that visualizes internal organs with a much finer resolution than conventional acoustic-resolution photoacoustic endoscopy systems. By combining gradient index (GRIN) lens-based optical focusing and ultrasonic ring transducer-based acoustic focusing, we achieved a transverse resolution as fine as ~10 μm at an optical working distance of 6.5 mm. The OR-PAEM system’s high-resolution intravital imaging capability is demonstrated through animal experiments.

Super-resolution microscopy techniques - capable of overcoming the diffraction limit of light - have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.

Photoacoustic microscopy (PAM) has been shown to be a valuable tool for quantifying hemoglobin oxygenation within single vessels. Recently, optical-resolution PAM was developed to achieve higher resolution by reducing the laser beam diameter, which increased the light intensity. As intensity increases, saturation of the optical absorption and multiphoton/ multi-step absorption can occur, which, together with the temperature dependence of thermal expansion, result in a non-linear dependence of the photoacoustic signal on the excitation pulse fluence. For hemoglobin, the major absorber in tissue for photoacoustic imaging, these non-linear phenomena have strong wavelength dependence. To enable quantitative photoacoustic measurements at different wavelengths in the presence of nonlinearity, a careful wide range analysis of the intensity-dependent absorption is required. Here, we built a photoacoustic spectrometer, using a tunable nanosecond optical parametric oscillator that operates between 410 nm and 2400 nm as our light source. To reduce uncertainty in our measurements due to inhomogeneous spatial distribution of the optical fluence, we used a flat-top beam illumination and a flat transducer which was mounted in reflection mode, effectively reducing quantitative measurements to a one dimensional problem. Intensity-dependent non-linear spectra of the photoacoustic signals of oxyand deoxy-hemoglobin were obtained. These measurements have the potential to contribute significantly to quantitative functional PAM.

Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed, high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per second with repeated two-dimensional raster scans of 100 by 50 points. The corresponding two-dimensional B-scan (50 A-lines) frame rate was 1800 Hz, and the one-dimensional A-scan rate was 90,000 Hz. The lateral resolution is 0.23±0.03 μm for Au nano-wire imaging, which is 2.0 times below the diffraction limit.

Recently, we developed a reflection-mode relaxation photoacoustic microscope, based on saturation intensity, to measure picosecond relaxation times using a nanosecond laser. Here, using the different relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, the oxygen saturation was quantified in vivo with single-wavelength photoacoustic microscopy. All previous functional photoacoustic microscopy measurements required imaging with multiple laser-wavelength measurements to quantify oxygen saturation. Eliminating the need for multi-wavelength measurements removes the influence of spectral properties on oxygenation calculations and improves the portability and cost-effectiveness of functional or molecular photoacoustic microscopy.

Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed, high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per second with repeated two-dimensional raster scans of 100 by 50 points. The corresponding twodimensional B-scan (50 A-lines) frame rate was 1800 Hz, and the one-dimensional A-scan rate was 90,000 Hz. The lateral resolution is 0.23±0.03 μm for Au nano-wire imaging, which is 2.0 times below the diffraction limit.

Picosecond absorption relaxation—central to many disciplines—is typically measured by ultrafast (femtosecond or picosecond) pump-probe techniques, which however are restricted to optically thin and weakly scattering materials or require artificial sample preparation. Here, we developed a reflection-mode relaxation photoacoustic microscope based on a nanosecond laser and measured picosecond absorption relaxation times. The relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, were measured at 576 nm. The added advantages in dispersion susceptibility, laser-wavelength availability, reflection sensing, and expense foster the study of natural—including strongly scattering and nonfluorescent—materials.

Improvements in the power level of sources near 1550 nm and in the efficiency of waveguide frequency doublers enabled us to lock a frequency-doubled source directly to the 5S(1/2) ? 5D(5/2) two-photon transitions near 778 nm. We obtained a sufficiently powerful second-harmonic signal, exceeding 2 mW, by doubling an external-cavity diode laser that was amplified by an erbium-doped fiber amplifier in a periodically poled LiNbO(3) channel waveguide. Our experimental scheme can be used for realizing compact, high-performance frequency standards near 1550 nm for fiber-optic communication and sensing applications.