This PDF file contains the front matter associated with SPIE Proceedings Volume 10504, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as a fuel source. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ_2) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ_2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance. It remains unclear whether NAD(P)H FLIM is sensitive to potential fates of glucose other than the TCA cycle and electron transport chain. We show that the lifetime of protein-bound NAD(P)H (τ_2) can distinguish the relative concentrations of malate dehydrogenase (TCA cycle enzyme) and lactate dehydrogenase (diverts carbon to/from lactate production) in solutions containing both enzymes. In cells, NAD(P)H FLIM was also sensitive to alterations in the path of carbon from glucose uptake to mitochondrial activity. Additionally, τ_2 was found to be the FLIM parameter best-suited for detecting these carbon-diverting shifts in cell metabolism, which may support mechanisms of drug resistance.

All blood vessels are lined with a single layer of endothelia cells which play a vital role in controlling the vessels in terms of blood flow, angiogenesis, vascular remodelling, response to pressure changes and blood borne chemical markers. Traditionally such responses to stimuli are examined in isolated cells, in a vascular preparation in which the vessels are opened up to lie flat or using relatively slow confocal or non-linear microscopy from the outside. We have developed a miniature probe that enables high speed, widefield imaging from within an intact pressurised vessel. This presentation will discuss the development of a 750 micron diameter probe which views orthogonally to the main optical axis to provide sub-cellular resolution images of around 300 cells in an intact, curved artery with the ability to rapidly change focus. Results of the imaging performance will be presented along with the biological context illustrating how this novel imaging modality when coupled with computational modelling enabled a new insight into the biological signalling processes within an intact vessel. By combining this new imaging system with a novel image processing pipeline results will be presented illustrating that the response to certain agonists is affected by pressure and the changing shape of the cells controls this response during a pressure rise. The work illustrates the way that an interdisciplinary approach bringing together novel optics, image processing, computational simulation and biology can lead to insights in the life sciences.

The cell cycle is extensively characterized, yet there is much to learn about the decision-making process involved in cell division. Here, the optical redox ratio (ratio of NADH to FAD fluorescence intensity) of MCF10A cells was imaged every 20 minutes over 12 hours using multiphoton microscopy. Cell tracking was used to monitor individual cells over time. We found a positive correlation in the variations of the optical redox ratio with the phases of the entire cell cycle. This study reveals the novel role of redox signaling in the progression the cell cycle.

Epithelial breast cells, which are involved in the production of milk fats, display altered lipid metabolism when they grow more malignant. At the same time, breast cancer cells show a higher degree of glycolysis, a mechanism to meet an increased demand for biomass as cells proliferate. Somewhat counterintuitively, breast cancer cells exhibit lower amounts of stored lipid, while glycolysis rates are up. We have investigated the kinetics of glycolysis, lipid synthesis, and lipid consumption rates in healthy and diseased epithelial breast cells. Using stimulated Raman scattering (SRS) microscopy of deuterated precursors, the cellular chemistry can be followed in space and time, revealing a clear link between glycolytic rate and the kinetics of lipid metabolism.

Fluorescence photo-switching of native, unmodified DNA using visible light enables label-free, nanoscale, single-molecule photon localization microscopy (PLM) of chromatin structure. Compared with conventional label-based super resolution imaging techniques, the label-free DNA-PLM has the advantage of faithfully resolving the native nucleotides under non-perturbing conditions, thus allowing a reliable analysis of the chromatin organization. Recently, we have developed an algorithm to quantify the chromatin spatial distribution based on label-free DNA-PLM images by calculating the fractal dimension from the chromatin cluster size and the number of photon emission events. For demonstration, we employed label-free DNA-PLM with TIRF illumination, and imaged the nuclei of ovarian cancer cells with three descending chromatin heterogeneities: the P53 mutation (M248), the wild type (A2780), and the wild type treated with a commonly-used chemotherapeutic drug celecoxib (Cele). Using the algorithm, we extracted the fractal dimensions for nuclear chromatin. We found that the fractal dimension is between 2 to 3 for all cells, which lies in the range of reported values from other techniques (e.g., TEM). We also observed that M248 has the highest fractal dimension while Cele has the lowest, a perfect match with the experimental expectations. We believe this study can provide a new approach to quantify label-free super-resolution imaging of macromolecular structures and could contribute to our knowledge of native in-vitro nuclear chromatin configurations.

We report femtosecond Laser-induced Breakdown Spectroscopy (fs-LIBS) measurements on several amino acids (Serine, Glutamine, and Cysteine) and Albumin protein solutions mixed with Ficoll polysaccharide at different proportions. The goal is to assess the effects of a host matrix on the identification and spectral characterization of amino acids by fs-LIBS. fs-LIBS utilizes an intense short laser pulse to obliterate a sample into basic constituents and to record the emission spectrum of atoms, ions, and molecules in the cooling down of the plasma plume. Several spectral peaks associated primarily with elemental composition of a sample were observed in the fs-LIBS spectra in a range from 200 to 950 nm. In addition, some molecular information associated with diatomic vibrational modes in certain molecules such as C-C and C-N were also obtained. The presence of Ficoll affects the relative intensity and broadening of the CN band, which could be considered as signatures of the amino acids. The fs-LIBS data and their analysis compare favorably with those derived from Fourier Transform Infrared Spectroscopy (FTIR). Interpretation of the spectral information enclosed in the emission of the diatomic molecules during laser ablation may lead to a better understanding of plume chemistry with a direct consequence on chemical analysis of complex samples such as amino acids. Altogether, the results demonstrate the potential of fs-LIBS technique as a detection method of biomolecules and for probing interactions of these biomolecules with a host matrix.

Tissue optical properties have become an increasingly promising avenue of diagnosis and screening for cancers and may provide contrast for real time monitoring of tumor ablation therapy. Of particular interest, are methods that can quantify scattering properties while providing spatial context or a map of the tissue being measured. Optical Coherence Tomography (OCT) is a non-destructive imaging modality with high 3D resolution which can be miniaturized into a probe compatible with common endoscopes. OCT has recently been used to quantify optical scattering properties, and endoscopic access to luminal organs allows examination of the thin epithelial layer, wherein many cancers originate. We present a fiber probe capable of quantifying optical properties with no distal optics providing low-cost disposable functionality. A reflection from the distal fiber face provides a common path reference through the fiber and eliminates the need for reference arm with dispersion compensation. A custom visible light OCT instrument was adapted to the self-reference fiber capable of a-scan imaging hundreds of microns into porcine esophagus tissue. B-scan images are produced by dragging the fiber along the tissue surface. Tissue was thermally ablated to create controllable scattering contrast with normal tissue. Image analysis with a custom MATLAB algorithm demonstrated significant increases in scattering coefficients which has been observed previously in a benchtop scanning OCT system. Visible light maximizes scatter contrast making Vis-OCT an ideal tool for cancer screening. Additionally, the lack of a distal optics and scanning mechanism offers a cost-effective, disposable functionality.

We present application of holographic cytology to image red blood cells in flow. We have previously shown that optical volume, the product of surface area and integrated optical phase, is an invariant parameter which can be measured using quantitative phase imaging, regardless of cell geometry or orientation, provided that the images are digitally refocused. In these experiments, we image cells in microfluidic channels to illustrate this invariance as well as provide new insights into mechanical cell properties.

Prostate-specific antigen (PSA) biomarker assays are the current clinical method for mass screening of prostate cancer. However, high false-positive rates are often reported due to PSA’s low specificity, leading to an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we demonstrated the feasibility of using cellular refractive indices (RI) as a unique contrast parameter to accomplish label-free detection of prostate cancer cells via variance testing, but were unable to determine if a specific cell was cancerous or noncancerous. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free imaging system, which allows for the detection of individual prostate cancer cells utilizing cellular RI as the only contrast parameter. Noncancerous prostate (BPH-1) cells and prostate cancer (PC-3) cells were mixed at varied ratios and measured concurrently. Additionally, we isolated and induced PC-3 cells to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors such as TGF-β1. The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced PC-3 cells as well as BPH-1 cells. EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. Our study shows promising clinical potential in utilizing the PC-TIR biosensor imaging system to not only detect prostate cancer cells, but also evaluate prostate cancer progression.

Antimicrobial resistance (AMR) has been recognized as an increasingly serious threat to the effective prevention and treatment of a wide range of infections caused by bacteria, parasites, viruses and fungi. Biofilms are of particular interest because up to 65% of microbial infections are associated with such microorganism growth. Unfortunately, biofilms are considered to be far more resistant to antimicrobial agents than planktonic cells due to the nature of a biofilm, which consists of a structured colony of bacteria embedded in a self-produced polymer matrix. However, the mechanisms of biofilm antibiotic resistance are not fully understood due to the complexity observed within a biofilm community and the lack of non-invasive biofilm examination techniques. Here, we present a novel approach, which will not only monitor the early stage of bacterial biofilm formation in real time in order to provide early warning against biofilm formation, but also provide a research and clinical toolbox for investigating biofilm antibiotic resistance. Our system employs guided mode resonance (GMR) imaging to monitor the earliest stage of biofilm formation on a silicon nitride substrate. GMR imaging works by tracking the resonant wavelength of a nano-structured substrate, which is extremely sensitive to local refractive index changes at the sensor surface to the extent that we can see the initial attachment of bacterial cells and formation of micro-colonies. We have also observed the formation of E. coli biofilms in clinically relevant concentrations as they occur, i.e. on a timescale of a few hours [1]. [1] Optica 4,2 229-234 (2017)

Intracellular dynamics are dominated by active transport driven by energetic processes far from equilibrium. Cytoskeletal restructuring, membrane motions and molecular motors use GTP and ATP to drive directed transport that is quasi-one-dimensional with speeds from 10 nm/sec to 10 microns/sec and persistence times tp as large as several seconds. Light scattering under these conditions can be in the lifetime-broadened Doppler shift regime as opposed to a random diffusive regime. The isotropic distribution of 1D transport within cells and tissues produces broad-band signatures that do not produce specific Doppler spectral peaks, but produce Doppler spectral edges that can be related to the mean squared speeds inside cells. The wDtp = 1 product provides a natural dividing line between the Doppler and the diffusive regimes, with a broad cross-over range into which many tissue-based light scattering processes fall. In this talk, I will show how the intracellular Doppler character of dynamic light scattering is derived and modeled, and provide experimental support from biodynamic imaging. Biodynamic imaging uses low-coherence digital holography to capture dynamic spectra in three dimensions from living tissue samples. Biodynamic imaging, based on changes in intracellular dynamics caused by applied therapeutics or changing environments, is expanding into multiple applications, including the selection of chemotherapy for personalized cancer care, screening of potential new therapeutics, and the selection of embryos for artificial reproductive technology. I will give an overview of these applications, describing how changes in biophysical behavior provide actionable biomarkers for clinical applications.

Light scattering by subcellular organelles and interfaces such as membranes can be utilized for quantitative measurement of cellular and tissue states. The structural information of the organelles can be inferred from light-scattering by analyzing the signal at a conjugate Fourier plane of a dark-field imaging system. Via implementation of Gabor filters on the Fourier plane, we can selectively allow only certain angles of scattering to pass during imaging. These scatter angles are related to the spatial frequencies of the scattering source. So in effect, the Gabor filters can be tuned to probe objects of certain size/shape and orientation. Based on this property, we had previously reported a morphometric parameter called Orientedness that can detect change in mitochondrial orientation during apoptosis. In this work, we present a subcellular segmentation technique to track Orientedness values over time in individual subcellular structures. This is achieved through a dynamic mask that changes its shape according to the shape of the organelles while they undergo chemically-induced morphological changes. The mask is generated from the Gabor-filtered images of the cell. We also propose a modification of the original Orientedness calculation using local-energy information. We demonstrate our method by tracking the morphology of subcellular structures within cells which have been overloaded with calcium. Our results show changes in the morphology of the subcellular structure in calcium-treated cells but not in the untreated control. Moreover a decrease in the aspect ratio and orientedness of the analyzed structures correlates with the onset of mitochondrial rounding and fission as seen in fluorescence. Together, our results suggest that light scattering based labelfree analysis of organelle structures may be used to track subcellular activity over time.

We labeled poly(vinyl) alcohol (PVA, M_w ≈ 85 kDa) linear chains with 5-([4,6-Dichlorotriazin-2-yl]amino) fluorescein hydrochloride and measured their translational diffusion within similar non-fluorescent PVA solutions using fluorescence correlation spectroscopy (FCS). We found that the measured correlation functions could be readily fit with an expression derived for a freely diffusive nanoprobe, allowing us to determine changes of apparent diffusion coefficients with changes of PVA concentration. The data indicate slowing down of the diffusion of the fluorescent PVA as the surrounding PVA concentration is increased. However, the changes of the diffusion of the labeled chains cannot be accounted for by corresponding changes of solution viscosity. Instead, we use an entropic-based model suggested by de Gennes and his collaborators, and fit the data with a stretched exponential [exp(-αcⁿ )] with c denoting the PVA concentration, n being related to the solvent quality, and α being a prefactor that is predicted to depend on probe size. We determined n = 0.75, suggesting that the host solvent, water, is a good solvent for the PVA as this value is close to the theoretical value n = 3/4. This result is similar to other measurements for different nanoprobes such as rhodamine 6G (n = 0.77) and phycoerythrin (n = 0.84), as previously reported (Michelman-Ribeiro, A., et al., Biomacromolecules 8, 1595-1600 (2007)). Further, we tested the α-dependence on probe size by collecting FCS data for various other nanoprobes, and found a systematic linear increase with size for globular nanoprobes which, however, differs from that of the PVA chains. That is, the hydrodynamic diameter of the non-globular, linear PVA chains appears not to be the determinant size for their diffusion within the polymeric PVA solution, indicating possible structural reconfiguration of the flexible, moving chains. These results are consistent with the proposed entropic-based model devised by de Gennes et al., and demonstrate how diffusing nanoprobes –globular or linear- can probe the quality of a host polymeric system.

The biomechanics of living tissues are critical to normal tissue function and disturbances in these properties are widely implicated in aging and disease. Protein fibres of the extracellular matrix (collagen and elastin) are the fundamental mechanical structures in connective tissues such as bone, cartilage and vasculature. We applied Brillouin light scattering (BLS) spectroscopy and quasistatic stress-strain testing to the study of the mechanics and structure of collagen and elastin fibres purified from connective tissues. BLS probes mechanical properties on a microscopic scale in biological tissues and thereby providing insights into structure-function relationships under normal and pathological conditions. The sensitivity of BLS measurements to fibre structure and hydration was investigated using samples mounted onto reflective substrates. We obtained a complete characterization of the mechanical tensor and elastic moduli which could be compared with complementary data from quasistatic stress-strain measurements at different hydration levels, hence giving the full description of fibre viscoelasticity.

Native fluorescence spectra play important roles in cancer detection. It is widely acknowledged that the emission spectrum of a tissue is a superposition of spectra of various salient fluorophores. However, component quantification is essentially an ill-posed problem. To address this problem, the native fluorescence spectra of normal human very low (LNCap), moderately metastatic (DU-145), and advanced metastatic (PC-3) cell lines were studied by the selected wavelength of 300 nm to investigate the key fluorescent molecules such as tryptophan, collagen and NADH. The native fluorescence spectra of cancer cell lines at different risk levels were analyzed using various machine learning algorithms for feature detection and develop criteria to separate the three types of cells. Principal component analysis (PCA), nonnegative matrix factorization (NMF), and partial least squares fitting were used separately to reduce dimension, extract features and detect biomolecular alterations reflected in the spectra. The scores corresponding to the basis spectra were used for classification. A linear support vector machine (SVM) was used to classify the spectra of the cells with different metastatic ability. In detection of signals coming from tryptophan and NADH with observed data corrupted by noise and inference, a sufficient statistic can be obtained based on the basis spectra retrieved using nonnegative matrix factorization. This work shows changes of relative contents of tryptophan and NADH obtained from native fluorescence spectroscopy may present potential criteria for detecting cancer cell lines of different metastatic ability.

We demonstrate a new 512x16 single photon avalanche diode (SPAD) based line sensor with per-pixel TCSPC histogramming for time-resolved, time-zoomable, FRET spectroscopy. The line sensor can operate in single photon counting (SPC) mode as well as time-correlated single photon counting (TCSPC) and per-pixel histogramming modes. TCSPC has been the preferred method for fluorescence lifetime measurements due to its collection of full decays as a histogram of arrival times. However, TCSPC is slow due to only capturing one photon per exposure and large timestamp data transfer requirements for offline histogramming. On-chip histogramming improves the data rate by allowing multiple SPAD pulses (up to one pulse per laser period) to be processed in each exposure cycle, along with secondly reducing the I/O bottleneck as only the final histogram is transferred. This can enable 50x higher acquisition rates (up to 10 billion counts per second), along with time-zoomable histogramming operation from 1.6ns to 205ns with 50ps resolution. A broad spectral range can be interrogated with the sensor (450-900nm). Overall, these sensors provide a unique combination of light sensing capabilities for use in high speed, sensitive, optical instrumentation in the time/wavelength domain. We test the sensor performance by observation of fluorescence resonance energy transfer (FRET) between FAM and TAMRA and between EGFP and RFP FRET standards.