Introduction to SPIE Photonics West BiOS conference 11658: Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XVIII

Powerful detection schemes in bioanalytics are associated with molecular specificity, high sensitivity, high spatial resolution and fast detection times. Within this presentation we will demonstrate that surface enhanced Raman spectroscopy (SERS) is able to meet all those requirements. We start with introducing various SERS detection schemes (direct SERS, SERS labels, molecular sensors) to identify bacterial infections or for the detection of atherosclerotic plaques. At the end we will present a tip enhanced Raman approach (TERS) to identify nanometer small virus particles. In order to further enhance the detection limit a novel coherent anti-Stokes Raman scattering approach using tip-enhanced techniques will be introduced.

Powerful detection schemes in bioanalytics are associated with the requirements for molecular specificity, high sensitivity and fast detection times. Surface enhanced Raman spectroscopy (SERS) is known to meet those requirements and the strong capability of this method in bioanalytical detection schemes is due to the enhancement of the molecular specific Raman fingerprint by 6 to 8 orders of magnitude. In order to increase the spatial resolution to the nanometer scale, SERS is combined with scanning probe microscopy creating tip enhanced Raman spectroscopy (TERS). Within this presentation, a literature review on SERS and TERS in bioanalytics is given.

Confocal scanners are used to scan through a large sample depth necessary in both high throughput and high content screening applications. However, this scanning takes time and increases the cost and optical complexity of medical diagnostic devices. Here we present extended depth-of-field engineered point spread function (ePSF) technology, combined with our universal modular subsystem SPINDLE®, to enable precision extended-depth imaging that can be combined with high-content analysis (HCA), without the need for z scanning, and without trading off light or lateral resolution.

Actin dynamics plays an important role in cell activities and drives a large range of cellular process such as cell division and cell motility. To visualize the actin dynamics, we introduce non-bleaching nanoscale imaging to capture actin dynamics over long time scales. Here, we demonstrate the high resolution quantification of actin dynamics by non-bleaching nanoscale imaging for the purpose of revealing resulting mechanical properties. Through this method, we visualized and quantified actin twisting dynamics through disassembly process by actin binding proteins.

Because the axial resolution of an optical microscope is in the order of the wavelength of light, imaging a nano-scale object is very challenging. Many nanometer-sectioning imaging technics such as total internal reflection fluorescence (TIRF) microscopy and metal-induced energy transfer (MIET) imaging have been invented to overcome this limitation. However, the measurement ranges of these methods along the axial direction are too short to cover even a single cell. Here we propose a new long-measurement-range nanometer-sectioning imaging scheme by using MIET, focal plane shifting, and sophisticated signal analysis. We have verified the principle and the feasibility of our proposed method by using an artificial sample; it is shown that the axial measurement range is extended from 100 nm to 500 nm.

Fluorescence detection is a well-established method for spectroscopy and sensing. However, since dye molecules are dipolar light sources, a large fraction of the emitted photons can be lost. An effective approach to overcome this problem relies on a planar antenna configuration, which beams the radiation pattern of the dye into a narrow cone. A planar antenna works like a Yagi-Uda antenna, but reflector and director elements are made of thin metal films. Here, by introducing a scanning optical fiber, which incorporates the reflector or the director, we demonstrate a tunable planar antenna for spectroscopic and sensing applications. Our results show that the radiation pattern narrows down to 26 degrees (FWHM), which implies a high collection efficiency by low-NA optics.

Improving the time resolution and sensitivity of Silicon-based Single Photon Avalanche Photodetectors (Si-SPAD) across the entire visible spectrum is critical to improve image quality in biomedical imaging applications such as positron emission tomography or fluorescence lifetime imaging. This work reports on the feasibility of manipulating the penetration depth of photons with 450 nm wavelengths to enhance absorption in Si-SPAD by means of photon trapping structures. Optical-electrical simulations suggest light can be directed towards critical regions of the semiconductor increasing the absorption from 54 to 90% with only 1.2μm of silicon and enhancing the probability of avalanche by electrons that leads to higher multiplication gain and speed of operation.

The sensing of neurotransmitters is currently a difficult task for scientists in the field of biomedical research. The design of a new compact and accurate sensor would benefit greatly the research being done on neurotransmitters. In this effect, optoelectronic sensors are ideal candidate as they are non-invasive and non-reactive to the test sample. This work describes the design, fabrication, calibration, and tests of a new type of neurotransmitter sensor using spectral analysis based on a Grism. This new optical sensor was fitted with a self calibrating algorithm and showed superior resolution as well as a smaller footprint than previously observed.

We report a localized surface plasmon resonance (LSPR) sensor that consists of noble metal and ferromagnetic material. This hybrid system offers tunable LSPR frequencies by the refractive index change of the surrounding medium as well as the external magnetic field around the nanostructures. We used a gold nanoparticle with Co and Fe. The thin ferromagnetic layers coated on gold nanoparticles have a linear dependence between relative permittivity with an applied magnetic field. The underlying mechanism is the change of the dielectric constant of the ferromagnetic film when it has aligned magnetization in the same direction of the external magnetic field. Consequently, change of the LSPR absorption spectrum of the hybrid nanostructures.

In the development of label-free and real-time optical biosensors, a straightforward and low-cost fabrication of the transducer is important, because it reduces costs and the complexity of the process. One possibility for such easy fabrication is electrospinning. It is a versatile and well-developed technique that allows the manufacturing of layers of nanofibers at a low cost with a Fabry-Pérot cavity-based optical response when illuminated with visible light. In this research, the suitability of such layers for the development of label-free and real-time optical biosensors is studied. For such aim, the nanofibers were biofunctionalized in flow with antibodies against bovine serum albumin through an intermediate layer of protein A/G. Then, BSA was flowed at a concentration of 10 µg/ml as the target analyte. As a result, biofunctionalization and biodetection processes were optically monitored in real-time successfully, demonstrating the suitability of such a simple-to-fabricate transducer for the development of label-free and real-time optical biosensors. Furthermore, in comparison with other optical transducers that require complex nanofabrication techniques, electrospun nanofibers can be deposited over vast areas to create several transducers in a single batch and at a low cost.

Microlasers have emerged as a promising approach for the detection or identification of different biomolecules. Most lasers were designed to reflect changes of molecular concentration within the cavity, without being able to characterize biophysical changes in the gain medium. Here, we report a strategy to extract and amplify polarized laser emissions from small molecules and demonstrate how molecular rotation interplays with lasing at the nanoscale. The concept of molecular lasing polarization was proposed and was first evidenced to increase accordingly as the fluorophore binds to larger biomolecules in a microcavity. By detecting the molecular rotational correlation time through stimulated emission, small molecules could be distinguished while conventional fluorescence polarization cannot. Theoretical models were developed to elucidate the underlying mechanisms. Finally, different types of small molecules were analyzed by adopting a Fabry-Pérot optofluidic laser. The results suggest an entirely new tool to quantify small molecules and guidance for laser emissions to characterize biophysical properties down to the molecular level.

The coupling of optical near fields and far-field scattering by arrays of nanoparticles can be harnessed to improve superresolution imaging, sub-diffraction limit beam focusing, or specialized sensing applications. Numerical simulation and optimization of all these processes commonly entails calculating far-field electric field distributions. However, widelyused simulation techniques such as finite difference time domain (FDTD), Mie theory, and the discrete dipole approximation (DDA) are computationally intensive for large numbers of particles and consequently restrict the size of the domain. Alternatively, the angular spectrum method (ASM) combined with a thin-object approximation, which are commonly used in lens-free holography and other applications to reconstruct images, are well-suited for computationallyefficient large-area calculations. But this approach is not necessarily accurate for nanostructured surfaces. Here we investigate the accuracy of the ASM in modeling the scattered field from a plane wave incident on a plane of randomly assembled nanoparticles. Many super-resolution, sub-diffraction limit, or specialized sensing applications utilize randomly distributed nanoparticles for the ease of placement. We investigate the dipole matched transmission model (DMT) using ASM for polystyrene and gold nanoparticles 30 nm, 60 nm, and 100 nm in diameter for various fill fractions of the nanoparticle plane. We compare the results from the ASM with DDA, which is validated against Mie theory calculations.

Monitoring the concentration of singlet oxygen and other free radicals in both intracellular and extracellular environment is of paramount importance, especially during therapy e.g., photo-dynamic, cold-plasma and radiation therapy. Singlet oxygen is one of the most cytotoxic reactive oxygen species and its intracellular measurement is challenging, particularly due to its short lifetime. Here we present poly-lactic-co-glycolic acid and polyacrylamide-based nanoparticles, with hydrophobic core, containing singlet-oxygen-sensor-green (SOSG), a membrane impermeable fluorescent dye, for measuring singlet oxygen in both aqueous phase and inside mammalian cells. The increase in fluorescence intensity of the dye at ~530 nm is directly proportional to the singlet oxygen concentration. The small size of the nanosensors (50–70nm) enables efficient cellular uptake, thus facilitating intracellular measurement of singlet oxygen using fluorescence microscopy. The polyacryamide nanosensor were used for monitoring intracellular singlet oxygen during plasma therapy, using room temperature helium plasma. Plasma sources produce a range of free radicals and has been utilized for various bio-medical applications, however its interaction with mammalian cells is not well studied. We characterize the changes in intracellular oxidative and toxicity till the cells become necrotic. These nanosensors provides an insight into the fundamental interaction of plasma with cells, thereby aiding in optimizing the therapeutic process.

We have investigated the effect of metal induced energy transfer from gold nanoaperture to fluorescence lifetime. We changed the orientation and position of dipole inside various diameter of nanohole and calculated relative lifetime by COMSOL Multiphysics and MATLAB. For the experiment, confocal microscopy setup was customized with blue laser at 479 nm and the nanoaperture was fabricated by both focused ion beam and e-beam lithography. Also, HPTS fluorescent dye in PVA solution was deposited on the nanoapertures by spin coating method instead of water droplets to avoid the effect from the film side and improve the contrast of the lifetime image. While the diameter of nanoapertures changed from 70 to 200 nm with 50 and 100 nm height, the maximum value of fluorescence lifetime tended to be proportional to the diameter and height due to energy transfer between dipole and metal. The maximum lifetime was 1.8 ns at 200 nm diameter which corresponded with calculation results. However, the lifetime difference between two heights was not linear tendency. The lifetime reduction factor was of about almost 10 for a 70 nm nanohole, and 3 for a 200 nm nanohole which was a maximum of 3 in the case of droplet. It is possible to distinguish the size of the nanohole using the lifetime reduction even at a scale below the diffraction limit and applied to single biomolecule detection.

We have investigated the plasmonic effect of a gold thin film on the optical properties under a range of combinations of incident wavelengths, incident angles and polarization states, while assuming various film thicknesses. Theoretical calculation was performed with rigorous coupled-wave analysis based on the temperature-dependent Drude-Lorentz dispersion model. The calculation method considers the effects of absorption, which is converted to heat in a gold thin film and can affect material parameters such as permittivity. Experimentally, light absorption and field enhancement factor were directly measured using near-field scanning optical microscopy. We have also measured the near-field distribution and thermal effects in the gold thin film. Absorption and field enhancement experimentally measured using three incident wavelengths of 488, 532, and 721 nm for a thin gold film with thicknesses 20, 50, and 70 nm showed good agreement with calculated data. Also observed was the disparity between the incident angles that correspond to maximum absorption and highest field enhancement. The results can help understand the thermal effects on optical properties of plasmonic nanostructures for applications in biological imaging and sensing techniques.