This PDF file contains the front matter associated with SPIE Proceedings Volume 10075 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Speckle formation is a limiting factor when using coherent sources for imaging and sensing, but can provide useful information about the motion of an object. Illumination sources with tunable spatial coherence are therefore desirable as they can offer both speckled and speckle-free images. Efficient methods of coherence switching have been achieved with a solid-state degenerate laser, and here we demonstrate a semiconductor-based degenerate laser system that can be switched between a large number of mutually incoherent spatial modes and few-mode operation. Our system is designed around a semiconductor gain element, and overcomes barriers presented by previous low spatial coherence lasers. The gain medium is an electrically-pumped vertical external cavity surface emitting laser (VECSEL) with a large active area. The use of a degenerate external cavity enables either distributing the laser emission over a large (~1000) number of mutually incoherent spatial modes or concentrating emission to few modes by using a pinhole in the Fourier plane of the self-imaging cavity. To demonstrate the unique potential of spatial coherence switching for multimodal biomedical imaging, we use both low and high spatial coherence light generated by our VECSEL-based degenerate laser for imaging embryo heart function in Xenopus, an important animal model of heart disease. The low-coherence illumination is used for high-speed (100 frames per second) speckle-free imaging of dynamic heart structure, while the high-coherence emission is used for laser speckle contrast imaging of the blood flow.

Localized surface plasmon resonance (LSPR) arises from the interaction of light with noble metal nanoparticles, which induces a collective oscillation in the free electrons. The size and shape of the metallic nanostructure significantly impact LSPR frequency and strength. Nanoplasmonic sensor has become a recent research focus due to its significant signal enhancement and robust signal transduction measured by extinction spectroscopy, fluorescence, Raman scattering, and absorption spectroscopy. Dark-field microscopy, in contrast, reports the scattered photons after light-matter interactions. In this case, the nanoparticles can be understood as dipole radiators whose free electrons oscillate in concert. Coupled with spectroscopy, this platform allows the collection of plasmonically scattered spectra from gold nanoparticles. Plasmonic coupling between electron-beam lithography patterned gold nanodisks (AuND) and colloidal gold nanoparticles (AuNP) can change the plasmonic resonance of the original entities, and can be effectively studied by dark-field hyperspectral microscopy. Typically, a pronounced redshift can be observed when plasmonic coupling occurs. When these nano-entities are functionalized with interactive surface moieties, biochemistry and molecular processes can be studied. In this paper, we will present the capability of assessing the process of immobilizing streptavidin-functionalized AuNPs on an array of biotin-terminated AuNDs. By monitoring changes in the LSPR band of AuNDs, we are able to evaluate similar processes in other molecular systems. In addition, plasmon coupling induced scattering intensity variations can be measured by an electron-multiplied charge-coupled device camera for rapid in situ monitoring. This method can potentially be useful in studying dynamic biophysical and biochemical processes in situ.

DNA hybridization, where two single-stranded DNA molecules form duplex through sequence-specific interactions, is a fundamental biological process. To gain better understanding, sequence-specific detection of hybridization at the singlemolecule level has been instrumental and can find a wide variety of applications. Nanoporous gold nanoparticle (NPGNP) array chip features large specific surface area and high-density plasmonic field enhancement known as “hot-spots” that are attractive in nanoplasmonic sensor development. In this paper, we discuss results on detecting single-molecule DNA hybridization on functionalizing NPG-NP array chip with unique bio-recognition elements towards both high sensitivity and specificity.

Biolasers are an emerging technology for next generation biochemical detection and clinical applications. Progress has recently been made to achieve lasing from biomolecules and single living cells. Tissues, which consist of cells embedded in extracellular matrix, mimic more closely the actual complex biological environment in a living body and therefore are of more practical significance. Here, we developed a highly versatile tissue laser platform, in which tissues stained with fluorophores are sandwiched in a high-Q Fabry-Pérot microcavity. Distinct lasing emissions from muscle and adipose tissues stained respectively with fluorescein isothiocyanate (FITC) and boron-dipyrromethene (BODIPY), and hybrid muscle/adipose tissue with dual-staining were achieved with a threshold of only ~10 μJ/mm². Additionally, we investigated how tissue structure/geometry, tissue thickness, and staining dye concentration affect the tissue laser. It is further found that, despite large fluorescence spectral overlap between FITC and BODIPY in tissues, their lasing emissions could be clearly distinguished and controlled due to their narrow lasing bands and different lasing thresholds, thus enabling highly multiplexed detection. Our tissue laser platform can be broadly applicable to various types of tissues/diseases. It provides a new tool for a wide range of biological and biomedical applications, such as diagnostics/screening of tissues and identification/monitoring of biological transformations in tissue engineering.

Structural and biological origins of light scattering in cells and tissue are still poorly understood. We demonstrate how this problem might be addressed through the use of transmission electron microscopy (TEM). For biological samples, TEM image intensity is proportional to mass-density, and thus proportional to refractive index (RI). By calculating the autocorrelation function (ACF) of TEM image intensity of a thin-section of cells, we essentially maintain the nanoscale ACF of the 3D cellular RI distribution, given that the RI distribution is statistically isotropic. Using this nanoscale 3D RI ACF, we can simulate light scattering through biological samples, and thus guiding many optical techniques to quantify specific structures. In this work, we chose to use Partial Wave Spectroscopy (PWS) microscopy as a one of the nanoscale-sensitive optical techniques. Hela cells were prepared using standard protocol to preserve nanoscale ultrastructure, and a 50-nm slice was sectioned for TEM imaging at 6 nm resolution. The ACF was calculated for chromatin, and the PWS mean sigma was calculated by summing over the power spectral density in the visible light frequency of a random medium generated to match the ACF. A 1-µm slice adjacent to the 50-nm slice was sectioned for PWS measurement to guarantee identical chromatin structure. For 33 cells, we compared the calculated PWS mean sigma from TEM and the value measured directly, and obtained a strong correlation of 0.69. This example indicates the great potential of using TEM measured RI distribution to better understand the quantification of cellular nanostructure by optical methods.

Melanosome is an organelle for synthesis, storage and transport the melanin, a major intrinsic pigment. In retinal pigmented epithelium (RPE), it is generally accepted that melanosome plays a critical photoprotective role, and it has been shown that that loss of melanin from RPE could be an early event towards age-related macular degeneration (AMD). Meanwhile, melanosome is also the major contributor to the optical properties of RPE, due to its high refractive index and the strong optical absorption of melanin. Therefore, a characterization and understanding the optical properties of melanin is of great interest to relate the physical and chemical changes of melanosomes, and their fundamental roles in RPE-related retinal diseases such as AMD. Here, we present a theoretical study to characterize the full optical properties of melanosomes. We modeled melanosomes as uniformly melanin filled spheroids, based on their morphology under transmission electron microscopy. T-matrix method was used to simulate the wavelength dependent total scattering, backscattering, absorption cross sections, and anisotropy factor. We verified our simulation on backscattering cross section of melanosome by comparing optical coherence tomography taken in visible and NIR ranges. In addition, we studied the changes of the optical properties of melanosomes on melanin bleaching. The results suggested a spectroscopic mechanism for optical detection of melanin loss by inverse spectroscopic optical coherence tomography.

Human immunodeficiency virus (HIV-1) infection remains a major health problem despite the use of highly active antiretroviral therapy (HAART), which has greatly reduced mortality rates. Due to the unavailability of an effective vaccine or a treatment that would completely eradicate the virus, the quest for new and combination therapies continues. In this study we explored the influence of Low Level Laser Therapy (LLLT) in HIV-1 infected and uninfected cells. Literature reports LLLT as widely used to treat different medical conditions such as diabetic wounds, sports injuries and others. The technique involves exposure of cells or tissue to low levels of red and near infrared laser light. Both HIV infected and uninfected cells were laser irradiated at a wavelength of 640 nm with fluencies ranging from 2 to 10 J/cm² and cellular responses were assessed 24 hours post laser treatment. In our studies, laser therapy had no inhibitory effects in HIV-1 uninfected cells as was indicated by the cell morphology and proliferation results. However, laser irradiation enhanced cell apoptosis in HIV-1 infected cells as the laser fluencies increased. This led to further studies in which laser irradiation would be conducted in the presence of HAART to determine whether HAART would minimise the detrimental effects of laser irradiation in infected cells.

Many of the earliest structural changes associated with neoplasia occur on the micro and nanometer scale, and thus appear histologically normal. Our group has established Inverse Spectroscopic OCT (ISOCT), a spectral based technique to extract nanoscale sensitive metrics derived from the OCT signal. Thus, there is a need to model light transport through relatively large volumes (< 50 um^3) of media with nanoscale level resolution. Finite Difference Time Domain (FDTD) is an iterative approach which directly solves Maxwell’s equations to robustly estimate the electric and magnetic fields propagating through a sample. The sample’s refractive index for every spatial voxel and wavelength are specified upon a grid with voxel sizes on the order of λ/20, making it an ideal modelling technique for nanoscale structure analysis. Here, we utilize the FDTD technique to validate the nanoscale sensing ability of ISOCT. The use of FDTD for OCT modelling requires three components: calculating the source beam as it propagates through the optical system, computing the sample’s scattered field using FDTD, and finally propagating the scattered field back through the optical system. The principles of Fourier optics are employed to focus this interference field through a 4f optical system and onto the detector. Three-dimensional numerical samples are generated from a given refractive index correlation function with known parameters, and subsequent OCT images and mass density correlation function metrics are computed. We show that while the resolvability of the OCT image remains diffraction limited, spectral analysis allows nanoscale sensitive metrics to be extracted.

Lipopolysaccharide (LPS) is an amphiphilic lipoglycan that is the primary component of the outer membrane of Gramnegative bacteria. Classified as a pathogen associated molecular pattern (PAMPs), LPS is an essential biomarker for identifying pathogen serogroups. Structurally, LPS is comprised of a hydrophobic lipophilic domain that partitions into the outer membrane of Gram-negative bacteria. Previous work by our team explored biophysical interactions of LPS in supported lipid bilayer assemblies (sLBAs), and demonstrated LPS-induced hole formation in DOPC lipid bilayers. Here, we have incorporated cholesterol and sphingomyelin into sLBAs to evaluate the interaction of LPS in a more physiologically relevant system. The goal of this work was to determine whether increasing membrane complexity of sLBAs, and changing physiological parameters such as temperature, affects LPS-induced hole formation. Integrating cholesterol and sphingomyelin into sLBAs decreased LPS-induced hole formation at lower concentrations of LPS, and bacterial serotype contributed to differences in hole formation as a response to changes in temperature. We also investigated the possibility of LPS-induced hole formation in cellular systems using the cytokine response in both TLR4 (+)/(-) murine macrophages. LPS was presented to each cell line in murine serum, delipidated serum, and buffer (i.e. no serum), and the resulting cytokine levels were measured. Results indicate that the method of LPS presentation directly affects cellular cytokine expression. The two model systems presented in this study provide preliminary insight into the interactions of LPS in the host, and suggest the significance of amphiphile-carrier interactions in regulating host-pathogen biology during infection.

Optical trapping has emerged as an essential tool for manipulating single biological material and performing sophisticated spectroscopy analysis on individual cell. The optical trapping technique has been used to grab and immobilize cells from a tightly focused laser beam emitted through a high numerical aperture objective lens. Coupling optical trapping with other technologies is possible and allows stable sample trapping, while also facilitating molecular, chemical and spectroscopic analysis. For this reason, we are exploring laser trapping combined with laser spectroscopy as a potential non-invasive method of interrogating individual cells with a high degree of specificity in terms of information generated. Thus, for the delivery of as much pathological information as possible, we use a home-build optical trapping and spectroscopy system for real time probing human immunodeficiency virus (HIV-1) infected and uninfected single cells. Briefly, our experimental rig comprises an infrared continuous wave laser at 1064 nm with power output of 1.5 W, a 100X high numerical aperture oil-immersion microscope objective used to capture and immobilise individual cell samples as well as an excitation source. Spectroscopy spectral patterns obtained by the 1064 nm laser beam excitation provide information on HIV-1 infected and uninfected cells. We present these preliminary findings which may be valuable for the development of an HIV-1 point of care detection system.

techniques to measure changes of the translational diffusion times and the rotational diffusion times of two nanoprobes, Alexa488 and FITC-Ficoll, dispersed in aqueous Ficoll solutions at room temperature. Analysis of the data indicated that the lifetimes of the nanoprobes appeared to be unaltered by the Ficoll solutions. In contrast, the FCS functions of each nanoprobe, which demonstrated slowing down of diffusion due to Ficoll, were adequately fitted with the expression of a freely diffusing nanoparticle. Similarly, the FA data indicated that the rotational diffusion of both nanoprobes was slowed down. The changes of the diffusion times and the rotational times of both nanoprobes could not be accounted for, however, by the corresponding changes of the viscosity of the solutions. Instead, we applied the entropic model proposed by de-Gennes and his collaborators, and fitted each set of diffusion data with a stretched exponential [exp(- αcⁿ)] with n being related to the quality of the solvent. We determined n-values close to the value one for both nanoprobes and for both diffusions, suggesting a theta-like behavior of the solutions. However, the 􀁄-values for the translation of both nanoprobes were larger than the corresponding ones derived for their rotation, indicating dissimilar local entropic effects. Together with calculations, the present results confirmed the slowing down of the diffusion processes of the nanoprobes due to crowding and, more significantly, provided through the nanoprobes insight into entangled but flexible polymeric structures of the concentrated solutions.

Many life-relevant interaction energies are in IR range, and it is reasonable to believe that some biochemical reactions inside cells can results in emission of IR photons. Cells can use this emission for non-chemical and non-electrical signaling. Detecting weak infrared radiation from live cells is complicated because of strong thermal radiation background and absorption of radiation by tissues. A microfluidic device with live cells inside a vacuum cryogenic environment should suppress this background, and thereby permit observation of live cell auto-luminescence or signaling in the IR regime. One can make IR-transparent windows not emitting in this range, so only the cell and a small amount of liquid around it will emit infrared radiation. Currently mid-IR spectroscopy of single cells requires the use of a synchrotron source to measure absorption or reflection spectra. Decreasing of thermal radiation background will allow absorption and reflection spectroscopy of cells without using synchrotron light. Moreover, cell auto-luminescence can be directly measured. The complete absence of thermal background radiation for cryogenically cooled samples allows the use IR photon-sensitive detectors and obtaining single molecule sensitivity in IR photo-luminescence measurements. Due to low photon energies, photo-luminescence measurements will be non-distractive for pressures samples. The technique described here is based upon US patent 9366574.