This PDF file contains the front matter associated with SPIE Proceedings Volume 10888, including the Title Page, Copyright Information, Table of Contents, Author and Conference Committee lists.

Cellular viscoelasticity is a biomarker for cancer type and toxin exposure. Current standard methods for probing cellular stiffness are slow, laborious, and utilize complex or indirect detection. These limitations prevent effective study of changes to viscoelasticity over time as well as longitudinal study of single cells. To enable direct and non-contact measurement of stiffness, we developed a quantitative phase imaging (QPI) based method to directly measure mechanical displacement in living cells in response to static loading. We calculated mechanical parameters, including shear stiffness, to discriminate between different cancer types and cell types that were exposed to varied levels of environmental and pharmacological toxins. We also demonstrated a correlation between our shear stiffness parameter and disorder strength, a measure of cellular refractive index homogeneity acquired via a single QPI image, showing the feasibility of high-throughput, nondestructive mechanical measurements. Now, we compare our methods to atomic force microscopy (AFM), the gold standard for measuring cellular viscoelastic characteristics. We evaluate multiple breast cancer cell lines that are dosed with varying concentrations of cytochalasin B, an actin network-disrupting toxin. Each group is characterized by a commercial AFM to measure Young’s modulus and indentation stiffness. The same groups are analyzed using our QPI system to simultaneously measure shear stiffness and disorder strength. Relationships between all four measurements are analyzed to determine the correlation between the QPI derived parameters and those found using the commercial AFM, and to explore the feasibility of using QPI as a high-throughput alternative to AFM for measurements of cellular viscoelasticity.

The heterogeneity and dynamic nature of cancerous tumors, such as those seen in breast cancer, pose a unique challenge in determining treatment regimens. The use of zebrafish as an in vivo model of breast cancer provides a high-throughput model with the potential to screen for efficacious drugs on a patient-by-patient basis. In this study, we use two-photon microscopy to measure metabolic changes in zebrafish with xenografted breast cancer tumors before, during, and after treatment with the anti-cancer drug paclitaxel. We use this metabolic imaging data to evaluate the zebrafish as a robust in vivo model of breast cancer. Preliminary results suggest the xenograft tumors respond to treatment with paclitaxel at 48 hours post treatment, as demonstrated by significant changes in NAD(P)H fluorescence lifetimes.

Meiosis is the specialized form of cell division that produces haploid cells to enable sexual reproduction. Central to this process is recombination between homologous chromosomes, which gives rise to genetic diversity. Faithful meiotic chromosome segregation, and thus fertility, also depends on the formation of crossovers between each pair of chromosomes. The number and spacing of crossovers is tightly regulated, but the mechanisms that govern crossover patterning remain unclear. Previous work from our lab demonstrated that the synaptonemal complex (SC), a protein polymer that forms between homologous chromosomes during early meiosis, is a liquid crystal that arises through regulated phase separation. We are currently investigating the hypothesis that this medium might enable biochemical signals to diffuse along the interface between paired chromosomes to coordinate crossover patterning. We identified and characterized a family of four RING finger proteins (ZHP-1-4) in C. elegans that cooperate to promote and limit the number of crossovers during meiosis. Here, using in vivo imaging and fluorescence correlation spectroscopy (FCS), we show that ZHP-3 is mobile within the SC, even after accumulating at crossover sites. We corroborate this result by measuring fluorescence recovery after photobleaching (FRAP), and further show that this property of ZHP-3 is unique among several pro-crossover factors. Our results, together with the known interactions of ZHP-1-4, suggest that a reaction-diffusion system within the SC may pattern crossovers during meiosis.

Ultraviolet (UV) spectroscopy is a powerful tool for quantitative biochemical analysis, but its application to molecular imaging and microscopy has been limited. Here we describe our recent work on ultraviolet hyperspectral interferometric (UHI) microscopy, which leverages coherent detection of optical fields to overcome significant challenges associated with UV spectroscopy when applied to molecular imaging. We demonstrate that this method enables quantitative spectral analysis of important endogenous biomolecules with subcellular spatial resolution and sensitivity to nanometer-scaled structures for label-free molecular imaging of live cells.

The structures of neuroretinal cells are fundamentally important for their functions. Therefore, elastic light scattering spectroscopy (ELSS) is an attractive approach to characterize the structural properties of retina, particularly due to its superb structural sensitivities down to several tens of nanometers. However, ELSS in living human retina is largely unexplored due to the lack of imaging tools. Here, enabled by visible and near infrared optical coherence tomography (vnOCT), we provided a quantitative measurement of ELSS in several interested anotomical layers in living human retina. Their biophysical implications will be discussed. In addition, vnOCT provided capillary oximetry in human retina, another important measurement in understanding retinal biophysics and pathology.

We will discuss optical imaging of the cells inside the cochlea. We will describe recent results for imaging the hair cells through an optically thinned section of the cochlea bone. A combination of optical coherence tomography, two photon fluorescence microscopy and femtosecond laser ablation is used.

Alterations to nanoscale structures, lymphatics, and microvasculature are early hallmarks of neoplasia as well as a variety of other diseases. Unfortunately, nanoscale alterations and microvasculature function, such as oxygen saturation, cannot be probed by histology. Furthermore, properly evaluating lymphatic and microvasculature organization can be challenging with histological slices. Optical Coherence Tomography (OCT) offers a promising noninvasive solution to evaluating these biomarkers in 3D in vivo. OCT has shown the ability to provide 3D maps of vasculature with flow rate and blood oxygenation, as well as, lymphatic organization with a resolution on the order of 1-10 microns. Our group has established Inverse Spectroscopic OCT (ISOCT), which measures nanoscale mass density tissue fluctuations and can distinguish between histologically normal cancerous and noncancerous tissue. However, the most influential underlying assumption that allows the distinction between subdiffractional structural alterations in tissue is that the region of interest (ROI) includes a homogenous tissue type with similar scattering and absorption properties. Therefore, the highly absorbing blood and low scattering lymphatics must be excluded from analysis. Traditional OCT techniques to isolate vasculature and its spectra require timely repetitive scanning protocols, and the commonly utilized near infrared operating bandwidths require vessel-like filters to locate lymphatics. Herein we show how vasculature location and spectra can be extracted with a single visible OCT scan. Additionally, we demonstrate the high image contrast from visible OCT allows lymphatic location to be well defined. Finally, we show ultrastructural metrics fall within physiologically reasonable ranges after excluding vasculature and lymphatics from the ROI.

Microcirculation provides key functions including nutrition, waste disposal, immune related transport, thermoregulation and others. This paper will provide an overview of methods, including our own, to image the microcirculation in animals and humans. It will further explore the extraction and use of sub-voxel data for fundamental biological discovery and medical diagnosis.

Mitochondrial dynamics such as fission, fusion and movement can reveal the physiological status of neurons and, most importantly, serve as diagnostic measures for several neurodegenerative diseases. Traditionally fluorescent probes have been used to track mitochondrial dynamics in neurons. However, neurons show low transfection efficiency, presenting challenges for the use of genetically-encoded fluorescent markers. Alternatively, synthetic fluorescent dyes are shown to hinder mitochondrial motility. In addition, all types of fluorescent probes are subject to photo-bleaching which precludes imaging for longer periods of time. To circumvent these issues, we propose a light-scattering based label-free technique called Optical Scatter Imaging (OSI) that is sensitive to changes in morphology. In this work, we employ a previously reported label-free parameter to probe the change in the size of organelles such as mitochondria as they undergo fusion or fission in neurons. In addition, we present a technique to track organelle motion using kymographs obtained from the label-free images and compare them with those obtained from fluorescent images. We demonstrate that the label-free kymograph can track organelles such as mitochondria even after the sample is photo-bleached.

Water is critical for skin to function normally as a barrier to prevent moisture and heat loss from a body. Raman spectroscopy has high potential in skin hydration analysis as the measurement requires no contacts with the skin. However, traditional CCD based Raman spectrometer has limited performance in detecting high energy vibrations including CH and OH groups. This work reports a customized InGaAs based Raman spectrometer for probing high energy vibration bands. Chicken and pork skin samples were analyzed, and their Raman spectra were compared to other tissues such as fat, tendon, and muscle to determine the spectroscopic identities of CH and OH groups. These results indicate that water components are mostly unbounded in skin tissues. The results further suggest that muscle and tendon components are beneficial for storing water in skin tissue and possibly preventing skin dehydration.

In this paper, we propose a fluorescence detection system using a smartphone camera instead of photodiodes, which is widely used for fluorescence detection of polymerase chain reaction (PCR) chips. Along with the development of smartphones and open platforms, these cameras are superior to photodiodes in terms of performance, cost and size. This recently developed smartphone camera is applied to the fluorescence detection during the DNA amplification process. While the photodiode provides only an average brightness, the fluorescence distribution inside the PCR chips can also be investigated using the camera. Therefore, the cause of unsatisfactory experimental results as well as the degree of amplification of DNA can be determined. In addition to an example of investigating the chamber condition through photographed fluorescence, a curve of change in relative fluorescence intensity is presented according to the number of PCR cycles. The experimental results show that the proposed system can be used for real-time PCR systems.