This PDF file contains the front matter associated with SPIE Proceedings Volume 9328, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

We propose the use of a high-refractive index glass microsphere in combination with a conventional fluorescence microscope for imaging sub-cellular organelles and biomolecules. The microsphere is placed on a sample that is immersed in water, collects the near-field nano-features of the sample and generates a magnified virtual image in the farfield, which is recorded through a conventional water immersion objective. We first investigate the imaging capability of the microspheres on size-calibrated fluorescent micro-/nano-particles. The experimental results obtained from a microsphere with 60 μm in diameter demonstrate imaging capability of features of ~λ/7-size (λ is the wavelength) with a magnification factor of 5.4. The position of the virtual image, the field-of-view (FOV) of the microsphere and the magnification factor are studied by using microspheres with different sizes. Finite Element Method (FEM) simulations are performed, providing key insight into the imaging effect of the microspheres with super-resolution capability. Moreover, the distribution and complex shape of different sub-cellular organelles, like centrioles, mitochondria and chromosomes in the AML12 cell line, are imaged with help of the glass microspheres. Thereafter, the subcellular location of mitochondrial encoded proteins could be studied.

Besides the many advantages minimally invasive surgery offers, the surgeon suffers from the loss of information, visual and mechanical (haptic feedback). The latter is an important tool, which helps the surgeon to localize tissue abnormalities (benign vs. malign tissue). We are aiming to generate a reliable constitutive FE model of the organ describing its mechanical properties by employing multiple elastographic measurement techniques at different scales (cell, tissue, and organ). A silicon phantom has been generated for the purpose of testing the transfer of information (delivery and processing of data). The stress-strain curve was recorded and embedded in the FE Model (Arruda-Boyce). A 2D displacement map was experimentally obtained from the phantom, which was in good agreement with the FE simulation.

Multi-modal probes allow for flexible choice of imaging equipment when performing quenched-phosphorescence O2 measurements: one- or two-photon, PLIM or intensity-based ratiometric read-outs. Spectral and temporal (e.g. FLIMPLIM) discrimination can be used to image O₂ together with pH, Ca²⁺, mitochondrial membrane potential, cell death markers or cell/organelle specific markers. However, the main challenge of existing nanoparticle probes is their limited diffusion across thick (> 20-50 μm) 3D cell models such as tumor spheroids. Here, we present new class of polymeric nanoparticle probes having tunable size, charge, cell-penetrating ability, and reporter dyes. Being spectrally similar to the recently described MM2, PA2 and other O₂ probes, they are 5-10 times brighter, demonstrate improved ratiometric response and their surface chemistry can be easily modified. With cultures of 2D and 3D cell models (fibroblasts, PC12 aggregates, HCT116 human colon cancer spheroids) we found cell-specific staining by these probes. However, the efficient staining of model of interest can be tuned by changing number of positive and negative surface groups at nanoparticle, to allow most efficient loading. We also demonstrate how real-time monitoring of oxygenation can be used to select optimal spheroid production with low variability in size and high cell viability.

Reactive oxygen species (ROS) play an essential role in facilitating signal transduction processes within the cell and modulating the injuries. However, the generation of ROS is tightly controlled both spatially and temporally within the cell, making the study of ROS dynamics particularly difficult. This study present a novel protocol to quantify the dynamic of the mitochondrial superoxide as a precursor of reactive oxygen species. To regulate the mitochondrial superoxide level, metabolic perturbation was induced by administration of potassium cyanide (KCN). The presented method was able to monitor and measure the superoxide production rate over time. Our results demonstrated that the metabolic inhibitor, potassium cyanide (KCN) induced a significant increase in the rate of superoxide production in mitochondria of fetal pulmonary artery endothelial cells (FPAEC). Presented method sets the stage to study different ROS mediated injuries in vitro.

Macrophages play a key role in atherosclerotic plaque destabilization and rupture, since they accumulate large amounts of lipid through the uptake of modified lipoproteins which results in foam cell formation. Cholesterol efflux is the process of removing cholesterol from macrophages in the subintima of the vessel wall, and efflux mechanism in a cell is one of the critical issues for the prevention of cardiovascular diseases. High density lipoproteins (HDL) stimulate cholesterol efflux from macrophage foam cells in the arterial wall. Radioisotope-labeled cholesterol analysis method is well known conventional method for observing cholesterol efflux. The major drawback of this method is its long and complicated process. Fluorescence intensity imaging schemes are replacing the radioisotope-labeled method in recent years for cholesterol efflux monitoring. Various spectroscopic methods are also adapted for cholesterol efflux imaging. Here we present a fluorescence lifetime imaging method for more quantitative observation of cholesterol efflux process in macrophages, which enables us to observe cholesterol level changes with various conditions. We used J774 macrophage cell and 25-NBD-cholesterol which is a famous cholesterol specific dye. Our lifetime imaging results clearly show cholesterol efflux rate very effectively. We believe that fluorescence lifetime analysis is new and very powerful for cholesterol imaging or monitoring.

A multi-wavelength (360nm – 440nm), real-time Photonic Cancer Detector (PCD) optical system based on GaN semiconductor laser technology is outlined. A proof of concept using blue laser technology for early detection of cancer has already been tested and proven for esophageal cancer. This concept is expanded to consider a wider range of wavelengths and the PCD will initially be used for early diagnosis of oral cancers. The PCD creates an image of the oral cavity (broad field white light detection) and maps within the oral cavity any suspicious lesions with high sensitivity using a narrow field tunable detector.

A polarization-sensitive hyperspectral imaging system (SkinSpect) has been built and evaluated using two-layer tissue phantoms, fabricated to mimic the optical properties of melanin in different epidermal thickness and hemoglobin in dermal layers. Multiple tissue-mimicking phantoms with varying top layer thicknesses were measured for optical system calibration and performance testing. Phantom properties were characterized and validated using SkinSpect. The resulting analysis shows that the proposed system is capable of distinguishing and differentiating the layer-dependent absorption spectra and the depths at which this absorption occurs.

Colorectal cancer is the second leading cause of cancer deaths in the United States, affecting more than 130,000 Americans every year1. Determining tumor margins prior to surgical resection is essential to providing optimal treatment and reducing recurrence rates. Colorectal cancer recurrence can occur in up to 20% of cases, commonly within three years after curative treatment. Typically, when colorectal cancers are resected, a margin of normal tissue on both sides of the tumor is required. The minimum margin required for colon cancer is 5 cm and for the lower rectum 2 cm. However, usually more normal tissue is taken on both sides of the tumor because the blood supply to the entire segment is removed with the surgery and therefore the entire segment must be removed. Anastomotic recurrences may result from inadequate margins. Pathologists look at the margins to ensure that there is no residual tumor and this is usually documented in the pathology report. We have developed a portable, point-of-care fiber bundle microendoscopy imaging system for detection of abnormalities in colonic epithelial microstructure. The system comprises a laptop, a modified fiber bundle image guide with a 1mm active area diameter and custom LabVIEW interface, and is approved for imaging surgically resected colon tissue at the University of Arkansas for Medical Sciences. The microendoscopy probe provides high-resolution images of superficial epithelial histology in real-time to assist surgical guidance and to localize occult regions of dysplasia which may not be visible. Microendoscopy images of freshly resected human colonic epithelium were acquired using the microendoscopy device and subsequently mosaicked using custom post-processing software. Architectural changes in the glands were mapped to histopathology H&E slides taken from the precise location of the microendoscopy images. Qualitatively, glandular distortion and placement of image guide was used to map normal and dysplastic areas of the colonic tumor and surrounding region from microendoscopy images to H&E slides. Quantitative metrics for correlating images were also explored and were obtained by analyzing glandular diameter and spatial distribution as well as image texture.

Cerenkov luminescence (CL) is generated when a charged particle moves faster than the speed of light in dielectric media. Recently CL imaging becomes an emerging technique with the use of radioisotopes. However, due to relatively weak blue light production and massive tissue attenuation, CL has not been applied widely. Therefore, we attempted to shift the CL emission to more near infrared (NIR) spectrum for better tissue penetration by using Cerenkov Radiation Energy Transfer (CRET). Gold nanoclusters were conjugated with NIR dye molecules (AuNc-IR820 and AuNc-ICG) to be activated with ultraviolet light. We found optimal conjugate concentrations of AuNc-NIR conjugates by spectroscopy system to generate maximal photon emission. When exposed by ultraviolet light, the emission of NIR light from the conjugates were verified. In quantitative analysis, AuNc-NIR conjugates emit brighter light signal than pure AuNc. This result implies that NIR fluorescent dyes (both IR820 and ICG) can be excited by the emission from AuNc. Following the above baseline experiment, we mixed F-18 fluorodeoxyglucose (F-18 FDG) radioisotope to the AuNc- NIR conjugates, to confirm NIR emission induced from Cerenkov radiation. Long pass filter was used to block Cerenkov luminescence and to collect the emission from AuNc-NIR conjugates. Instead of one long exposure imaging with CCD, we used multiple frame scheme to eliminate gamma radiation strike in each frame prior to combination. In summary, we obtained NIR emission light from AuNc-NIR conjugated dyes that is induced from CL. We plan to perform in vivo small animal imaging with these conjugates to assess better tissue penetration.

Optoporation, the laser-induced transient cell membrane perforation, has emerged as a powerful non-invasive and highly efficient cell-transfection technique. It is usually done by targeting individual cells manually which significantly limits number of addressable cells. We present an experimental setup with custom-made software control for automated cell optoporation experiments. The automated software evaluates contrast edges in bright-field sample images to identify cell locations for laser illumination and controls the hardware to transiently laser-illuminate these positions. By software controlled stitching together of several microscopic field-of-views in principle all cells in a culture dish can be targeted without further user interaction. The software-based automation allows to significantly increase the number of treatable cells compared to a manual approach. We illustrate the experimental capabilities in CHO-cell optoporation experiments with highly focused beam-shaped sub-15 fs pulses.

There is a growing interest in label-free, optical techniques like digital holographic microscopy (DHM) and optical cell stretching, since the interaction with samples is minimized. Because optical manipulation strongly depends on the optical and physiological properties of the investigated material, we combined the usage of these methods for the characterization of pancreatic tumor cells. Our results demonstrate that cells of distinct differentiation levels, or different expression in only one protein, show differences in their deformability. Additionally, the DHM results showed only few variations in the refractive index, indicating that it does not significantly influence the results of the optical cell stretching. Thus, the combined usage of the two technologies represents a promising new approach for tumor cell characterization.

Protein microarrays are used various research areas including drug discovery, diagnosis, and analysis of protein-ligand interactions. Their efficacy depends on a well-defined pattern of immobilized proteins that also have retained their bioactivity. Protein microarrays are classically fabricated using the robotic spotting drop method (“pin printing”), which can lead to spots with uneven protein concentration within the spotted area, leading to difficult to quantify readings. Among the alternative techniques, microcontact printing (μCP) with a poly(dimethylsiloxane) (PDMS) stamp appears to deliver more defined protein patterns on surfaces, while maintaining bioactivity for a wide range of proteins. Here we have quantitatively compared the distribution of fluorescently labeled proteins deposited using direct pipetting, pin printing and μCP printing with flat stamps onto various functionalized glass surfaces of different contact angles through fluorescent microscopy. The uniformity of the deposited protein spots across deposition techniques was also qualitatively analyzed. It was found that with the use of either the direct pipetting or pin printing techniques that protein concentration on surfaces varied largely across surfaces with different contact angles, whereas adsorption did not vary significantly when using the μCP printing Furthermore, when μCP printing was performed with flat relief structures the spot inhomogeneity was lower than when classical methods were used, and even less so when a pyramid relief structure was used. This suggests that μCP printing with pyramid relief structures could produce protein patterns on various surfaces and with increased spot uniformity to enable more reliable protein microarrays.

Skin blood haemoglobin saturation (𝑠_b) can be estimated with hyperspectral imaging using the wavelength (λ) range of 450-700 nm where haemoglobin absorption displays distinct spectral characteristics. Depending on the image size and photon transport algorithm, computations may be demanding. Therefore, this work aims to evaluate subsets with a reduced number of wavelengths for 𝑠_b estimation. White Monte Carlo simulations are performed using a two-layered tissue model with discrete values for epidermal thickness (𝑇_epi) and the reduced scattering coefficient (μ'_s ), mimicking an imaging setup. A detected intensity look-up table is calculated for a range of model parameter values relevant to human skin, adding absorption effects in the post-processing. Skin model parameters, including absorbers, are; μ'_s (λ), 𝑇_epi, haemoglobin saturation (𝑠_b), tissue fraction blood (𝑐_b) and tissue fraction melanin (𝑐_mel). The skin model paired with the look-up table allow spectra to be calculated swiftly. Three inverse models with varying number of free parameters are evaluated: A(𝑠_b, 𝑐_b), B(𝑠_b, 𝑐_b, 𝑐_mel) and C(all parameters free). Fourteen wavelength candidates are selected by analysing the maximal spectral sensitivity to 𝑠_b and minimizing the sensitivity to 𝑐_b. All possible combinations of these candidates with three, four and 14 wavelengths, as well as the full spectral range, are evaluated for estimating 𝑠_b for 1000 randomly generated evaluation spectra. The results show that the simplified models A and B estimated 𝑠_b accurately using four wavelengths (mean error 2.2% for model B). If the number of wavelengths increased, the model complexity needed to be increased to avoid poor estimations.

Optical projection tomography (OPT) is a mesoscopic scale optical imaging technique for specimens between 1mm and 10mm. Although OPT is widely used for in vivo and ex vivo imaging, its applications in high intensity tissues such as bone and thick samples are limited due to the strong absorption of the light. In contrast, X-ray micro-CT is suitable for high intensity tissue imaging but its contrast of soft tissue is poor. Therefore, imaging tools with both strong penetration and high contrast are in great demand. To address this issue, we develop a dual-modality system integrating both OPT and micro-CT. In this paper, this dual-modality system is applied to dynamic imaging of a clearing process of a mouse paw. The clearing process is essential in OPT when imaging thick or intensity tissues since it can make high intensity tissues optically transparent. In our experiment, we scan the mouse paw with our system – before, during and after optical clearing. Each time we scan CT first and then the OPT. After acquisition, 3-dimentional volumes of OPT and CT are reconstructed separately. Then we use a rigid image registration algorithm to register these volumes. Finally, the volumes are merged together. The experimental results show our bimodal system performs better than single OPT or CT system when processing tissues with both high intensity and soft parts.

In the past decade, the efficacy of existing therapies and the discovery of innovative treatments for Central Nervous System (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. In order to better understand the fundamental mechanisms that regulate diseases of the CNS, a fast fluorescence hyperspectral imaging platform was designed to track simultaneously various neurotransmitter receptors trafficking in and out of synapses. With this hyperspectral imaging platform, it was possible to image simultaneously five different synaptic proteins, including subtypes of glutamate receptors (mGluR, NMDAR, AMPAR), postsynaptic density proteins, and signaling proteins. This new imaging platform allows fast simultaneous acquisitions of at least five fluorescent markers in living neurons with a high spatial resolution. This technique provides an effective method to observe several synaptic proteins at the same time, thus study how drugs for CNS impact the spatial dynamics of these proteins.

Connective tissue progenitors (CTPs) are defined as the heterogeneous population of tissue resident stem and progenitor cells capable of proliferating and differentiating into connective tissue phenotypes. The prevalence and variation in clonal progeny of CTPs can be characterized using a colony formation assay. However, colony assays do not directly assess the characteristics of the colony founding CTP. We developed a large field of view, time lapse microscopy system with phase contrast and fluorescence capabilities that enables tracking from seeding through colony formation.

Cells derived from the trabecular surface of bone were prepared and seeded in an Ibidi-Ph+ chamber slide. Phase contrast images of the slide were obtained every hour using a DMI6000 Leica microscope, 10X objective, and Retiga 2000R camera. Cells were stained using fluorescent antibodies for multiple markers at the time of plating to determine marker expression on seeded cells and re-stained to determine expression on their progeny. Colonies were identified and characterized using automated image processing and quantitative analysis methods. Following colony identification, the time lapse was reversed to identify and characterize the colony founding CTP according to morphology and marker expression. As a representative example, a CD73⁺/CD90^-/CD105^- and a CD73⁺/CD90⁺/CD105^- CTP resulted in a colony with an area of 3720826 microns² and percent area expression of 2.98%, 3.62%, and 1.13% for CD73, CD90, and CD105, respectively.

This method can be used to study CTPs and other stem and progenitor cell populations to benefit point-of-care methods for assay and isolation in cell based therapies.

Brillouin and Raman microspectroscopies are powerful techniques for noninvasive optical imaging. In particular, Brillouin microscopy is a unique technique, which allows assessing sample’s mechanical properties with optical level spatial resolution. Recent advances in background-free Brillouin microspectroscopy make it possible to image turbid samples without substantial degradation of the data quality. In this report, we applied Brillouin spectroscopy to imaging individual cells. These viscoelastic measurements were supplemented by chemically specific measurements using confocal Raman microspectroscopy.

Infrared (IR) imaging spectroscopy of human liver tissue slices has been used to identify and characterize liver metastasis of colorectal origin which was surgically removed from a consenting patient and frozen without formalin fixation or dehydration procedures, so that lipids and water remain in the tissues. First, a k-means clustering analysis, using metrics from the IR spectra, identified groups within the image. The groups were identified as tumor or nontumor regions by comparing to an H and E stain of the same sample after IR imaging. Then, calibrant IR spectra of protein, several fats, glycogen, and polyvinyl alcohol were isolated by differencing spectra from different regions or groups in the image space. Finally, inner products (or scores) of the IR spectra at each pixel in the image with each of the various calibrants were calculated showing how the calibrant molecules vary in tumor and nontumor regions. In this particular case, glycogen and protein changes enable separation of tumor and nontumor regions as shown with a contour plot of the glycogen scores versus the protein scores.

Bioluminescence tomography (BLT) is a powerful optical molecular imaging modality, which enables non-invasive realtime in vivo imaging as well as 3D quantitative analysis in preclinical studies. In order to solve the inverse problem and reconstruct inner light sources accurately, the prior structural information is commonly necessary and obtained from computed tomography or magnetic resonance imaging. This strategy requires expensive hybrid imaging system, complicated operation protocol and possible involvement of ionizing radiation. The overall robustness highly depends on the fusion accuracy between the optical and structural information.

In this study we present a pure optical bioluminescence tomographic system (POBTS) and a novel BLT method based on multi-view projection acquisition and 3D surface reconstruction. The POBTS acquired a sparse set of white light surface images and bioluminescent images of a mouse. Then the white light images were applied to an approximate surface model to generate a high quality textured 3D surface reconstruction of the mouse. After that we integrated multi-view luminescent images based on the previous reconstruction, and applied an algorithm to calibrate and quantify the surface luminescent flux in 3D.Finally, the internal bioluminescence source reconstruction was achieved with this prior information.

A BALB/C mouse with breast tumor of 4T1-fLuc cells mouse model were used to evaluate the performance of the new system and technique. Compared with the conventional hybrid optical-CT approach using the same inverse reconstruction method, the reconstruction accuracy of this technique was improved. The distance error between the actual and reconstructed internal source was decreased by 0.184 mm.

Over 30% of combat injuries involve peripheral nerve injury compared to only 3% in civilian trauma. In fact, nerve dysfunction is the second leading cause of long-term disability in injured service members and is present in 37% of upper limb injuries with disability. Identification and assessment of non-penetrating nerve injury in trauma patients could improve outcome and aid in therapeutic monitoring. We report the use of Raman spectroscopy as a noninvasive, non-destructive method for detection of nerve degeneration in intact nerves due to non-penetrating trauma. Nerve trauma was induced via compression and ischemia/reperfusion injury using a combat relevant swine tourniquet model (>3 hours ischemia). Control animals did not undergo compression/ischemia. Seven days post-operatively, sciatic and femoral nerves were harvested and fixed in formalin. Raman spectra of intact, peripheral nerves were collected using a fiber-optic probe with 3 mm diameter spot size and 785 nm excitation. Data was preprocessed, including fluorescence background subtraction, and Raman spectroscopic metrics were determined using custom peak fitting MATLAB® scripts. The abilities of bivariate and multivariate analysis methods to predict tissue state based on Raman spectroscopic metrics are compared. Injured nerves exhibited changes in Raman metrics indicative of 45% decreased myelin content and structural damage (p<<0.01). Axonal and myelin degeneration, cell death and digestion, and inflammation of nerve tissue samples were confirmed via histology. This study demonstrates the non-invasive ability of Raman spectroscopy to detect nerve degeneration associated with non-penetrating injury, relevant to neurapraxic and axonotmetic injuries; future experiments will further explore the clinical utility of Raman spectroscopy to recognize neural injury.

Fourier-transform imaging spectrometers are rapidly developed due to their extensive use in industrial monitoring, target detection, and chemical identification. Static Fourier-transform imaging spectrometer (SFIS) containing a birefringent interferometer is one of the most popular directions due to its inherent robustness. However, the SFIS suffers from its low achievable signal-to-noise ratio (SNR) because of the restriction of incident angle. Meanwhile, in applications, the SNR is perhaps the most important factor to determine the usefulness of an instrument. In this paper, we report here a Static Fourier-transform imaging spectrometer based on differential structure (SFIS-DS) in the 400-800nm wavelength range with a high SNR. As in electronic system, the differential structure can double optical efficiency and strongly restrain common mode error in the SFIS-DS. And the differential structure described here is also available for any instruments containing a birefringent interferometer. However, the drawback of the SFIS-DS is that the two images obtained by the two differential channels need precise registration which can be overcome by a sub-pixel spatial registration algorithm. The experimental results indicate the SFIS-DS can increase the SNR by no less than 40%.

A major limitation of spontaneous Raman scattering is its intrinsically weak signals, which makes Raman analysis or imaging of biological specimens slow and impractical for many applications. To address this, we report the development of a novel modulated multifocal detection scheme for simultaneous acquisition of full Raman spectra from a 2-D m × n multifocal array. A spatial light modulator (SLM), or a pair of galvo-mirrors, is used to generate m × n laser foci. Raman signals generated within each focus are projected simultaneously into a spectrometer and detected by a CCD camera. The system can resolve the Raman spectra with no crosstalk along the vertical pixels of the CCD camera, e.g., along the entrance slit of the spectrometer. However, there is significant overlap of the spectra in the horizontal pixel direction, e.g., along the dispersion direction. By modulating the excitation multifocal array (illumination modulation) or the emitted Raman signal array (detection modulation), the superimposed Raman spectra of different multifocal patterns are collected. The individual Raman spectrum from each focus is then retrieved from the superimposed spectra using a postacquisition data processing algorithm. This development leads to a significant improvement in the speed of acquiring Raman spectra. We discuss the application of this detection scheme for parallel analysis of individual cells with multifocus laser tweezers Raman spectroscopy (M-LTRS) and for rapid confocal hyperspectral Raman imaging.

Leukemia stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in the peripheral blood of leukemia patients. Since leukemic stem cells are also resistant to standard chemotherapeutic regimens, new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial targeting studies we utilized a bioinformatics approach to design an antibodyfluorescent nanoparticle conjugate for targeting to these leukemic stem cells and to minimize targeting to normal stemprogenitor cells.

Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell RS4;11 (with putative immunophenotype CD133+/CD24+/-, CD34+/-, CD38+, CD10-/Flt3+) was spiked into normal hematopoietic stem-progenitor cells obtained from a “buffy coat” prep (with putative immunophenotype CD133- /CD34+/CD38-/CD10-/Flt-3-) to be used as a model human leukemia patient. To analyze the model system, digital data mixtures of the two cell types were first created and assigned classifiers in order to create truth sets. ROC (Receiver Operating Characteristic) and multidimensional cluster analyses were used to evaluate the specificity and sensitivity of the immunophenotyping panel and for automated cell population identification, respectively. Costs of misclassification (false targeting) were also accounted for by this analysis scheme. Ultimately, this analysis scheme will be applied to use of nanoparticle-antibody conjugates at therapeutic doses for targeted killing of leukemia stem cells preferentially to normal stem –progenitor cells.

Laser-Induced Breakdown Spectroscopy (LIBS) and Raman Spectroscopy have rich histories in the analysis of a wide variety of samples in both in situ and remote configurations. Our team is working on building a deployable, integrated Raman and LIBS spectrometer (RLS) for the parallel elucidation of elemental and molecular signatures under Earth and Martian surface conditions. Herein, results from remote LIBS and Raman analysis of biological samples such as amino acids, small peptides, mono- and disaccharides, and nucleic acids acquired under terrestrial and Mars conditions are reported, giving rise to some interesting differences. A library of spectra and peaks of interest were compiled, and will be used to inform the analysis of more complex systems, such as large peptides, dried bacterial spores, and biofilms. These results will be presented and future applications will be discussed, including the assembly of a combined RLS spectroscopic system and stand-off detection in a variety of environments.

Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, “untargeted” imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

Oftentimes cells are removed from the body for disease diagnosis or cellular research. This typically requires fluorescent labeling followed by sorting with a flow cytometer; however, possible disruption of cellular function or even cell death due to the presence of the label can occur. This may be acceptable for ex vivo applications, but as cells are more frequently moving from the lab to the body, label-free methods of cell sorting are needed to eliminate these issues. This is especially true of the growing field of stem cell research where specialized cells are needed for treatments. Because differentiation processes are not completely efficient, cells must be sorted to eliminate any unwanted cells (i.e. un-differentiated or differentiated into an unwanted cell type). In order to perform label-free measurements, non-linear optics (NLO) have been increasingly utilized for single cell analysis because of their ability to not disrupt cellular function. An optical system was developed for the measurement of NLO in a microfluidic channel similar to a flow cytometer. In order to improve the excitation efficiency of NLO, a scanned Bessel beam was utilized to create a light-sheet across the channel. The system was tested by monitoring twophoton fluorescence from polystyrene microbeads of different sizes. Fluorescence intensity obtained from light-sheet measurements were significantly greater than measurements made using a static Gaussian beam. In addition, the increase in intensity from larger sized beads was more evident for the light-sheet system.

Introduction: The International Society for Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). The CytometryML schemas, are based in part upon the Flow Cytometry Standard and Digital Imaging and Communication (DICOM) standards. CytometryML has and will be extended and adapted to include MIFlowCyt, as well as to serve as a common standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). Individual major elements of the MIFlowCyt schema were translated into XML and filled with reasonable data. A small section of the code was formatted with HTML formatting elements. Results: The differences in the amount of detail to be recorded for 1) users of standard techniques including data analysts and 2) others, such as method and device creators, laboratory and other managers, engineers, and regulatory specialists required that separate data-types be created to describe the instrument configuration and components. A very substantial part of the MIFlowCyt element that describes the Experimental Overview part of the MIFlowCyt and substantial parts of several other major elements have been developed. Conclusions: The future use of structured XML tags and web technology should facilitate searching of experimental information, its presentation, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate in publications adherence to the MIFlowCyt standard. The use of CytometryML together with XML technology should also result in the textual and numeric data being published using web technology without any change in composition. Preliminary testing indicates that CytometryML XML pages can be directly formatted with the combination of HTML and CSS.

Tumor regions under hypoxic or low oxygen conditions respond less effectively to many treatment strategies, including radiation therapy. A novel investigational therapeutic, NVX-108 (NuvOx Pharma), has been developed to increase delivery of oxygen through the use of a nano-emulsion of dodecofluoropentane. By raising pO₂ levels prior to delivering radiation, treatment efficacy may be improved. To aid in evaluating the novel drug, oxygen tension was quantitatively measured, spatially and temporally, to record the effect of administrating NVX-108 in an orthotopic mammary window chamber mouse model of breast cancer. The oxygen tension was measured through the use of an oxygen-sensitive coating, comprised of phosphorescent platinum porphyrin dye embedded in a polystyrene matrix. The coating, applied to the surface of the coverslip of the window chamber through spin coating, is placed in contact with the mammary fat pad to record the oxygenation status of the surface tissue layer. Prior to implantation of the window chamber, a tumor is grown in the SCID mouse model by injection of MCF-7 cells into the mammary fat pad. Two-dimensional spatial distributions of the pO₂ levels were obtained through conversion of measured maps of phosphorescent lifetime. The resulting information on the spatial and temporal variation of the induced oxygen modulation could provide valuable insight into the optimal timing between administration of NVX-108 and radiation treatment to provide the most effective treatment outcome.

Cardiotoxicity is the major cause of drug withdrawal from the market, despite rigorous toxicity testing during the drug development process. Existing safety screening techniques, some of which are based on simplified cellular assays, others on electrical (impedance) or optical (fluorescent microscopy) measurements, are either too limited in throughput or offer too poor predictability of toxicity to be applied on large numbers of compounds in the early stage of drug development. We present a compact optical system for direct (label-free) monitoring of fast cellular movements that enable low cost and high throughput drug screening. Our system is based on a high-speed lens-free in-line holographic microscope. When compared to a conventional microscope, the system can combine adequate imaging resolution (5.5 μm pixel pitch) with a large field-of-view (63.4 mm²) and high speed (170 fps) to capture physical cell motion in real-time. This combination enables registration of cardiac contractility parameters such as cell contraction frequency, total duration, and rate and duration of both contraction and relaxation. The system also quantifies conduction velocity, which is challenging in existing techniques. Additionally, to complement the imaging hardware we have developed image processing software that extracts all the contractility parameters directly from the raw interference images. The system was tested with varying concentration of the drug verapamil and at 100 nM, showed a decrease in: contraction frequency (-23.3% ± 13%), total duration (-21% ± 5%), contraction duration (-19% ± 6%) and relaxation duration (-21% ± 8%). Moreover, contraction displacement ceased at higher concentrations.

Femtosecond laser microscopes have been used as both micro and nanosurgery tools. The optical knock-out of undesired cells in multiplex cell clusters shall be further reported on in this study.

Femtosecond laser-induced cell death is beneficial due to the reduced collateral side effects and therefore can be used to selectively destroy target cells within monolayers, as well as within 3D tissues, all the while preserving cells of interest. This is an important characteristic for the application in stem cell research and cancer treatment. Non-precise damage compromises the viability of neighboring cells by inducing side effects such as stress to the cells surrounding the target due to the changes in the microenvironment, resulting from both the laser and laser-exposed cells.

In this study, optimum laser parameters for optical cleaning by isolating single cells and cell colonies are exploited through the use of automated software control. Physiological equilibrium and cellular responses to the laser induced damages are also investigated. Cell death dependence on laser focus, determination and selectivity of intensity/dosage, controllable damage and cell recovery mechanisms are discussed.

In this paper, we discuss a new methodology based on lens-free imaging to perform wound healing assay with unprecedented statistics. Our video lens-free microscopy setup is a simple optical system featuring only a CMOS sensor and a semi coherent illumination system. Yet it is a powerful means for the real-time monitoring of cultivated cells. It presents several key advantages, e.g., integration into standard incubator, compatibility with standard cell culture protocol, simplicity and ease of use. It can perform the follow-up in a large field of view (25 mm²) of several crucial parameters during the culture of cells i.e. their motility, their proliferation rate or their death. Consequently the setup can gather large statistics both in space and time. But in the case of tissue growth experiments, the field of view of 25 mm² remains not sufficient and results can be biased depending on the position of the device with respect to the recipient of the cell culture. Hence, to conduct exhaustive wound healing assay, here we propose to enlarge the field of view up to 10 cm² through two different approaches. The first method consists in performing a scan of the cell culture by moving the source/sensor couple and then stitch the stack of images. The second is to make an acquisition by scanning with a line scan camera. The two approaches are compared in term of resolution, complexity and acquisition time. Next we have performed acquisitions of wound healing assay (keratinocytes HaCaT) both in real-time (25 mm²) and in final point (10 cm²) to assess the combination of these two complementary modalities. In the future, we aim at combining directly super wide field of view acquisitions (>10 cm²) with real time ability inside the incubator.

Cytometry has wide applications in biomedicine for cell differentiation or disease monitoring. Here we report our newly developed two dimensional (2D) light scattering static cytometric technique for single and multiple cell analysis. The static cytometer adopts a scanning fiber probe for cell excitation and obtains 2D light scattering patterns on a complementary metal oxide semiconductor (CMOS) detector. Our results show that experimental 2D light scattering patterns obtained from single yeast cells are with fringe structure while those from multiple yeast cells give speckle patterns. The experimental results compare favorably with our 2D light scattering Mie theory simulations for both single and multiple cells. The varying of 2D light scattering patterns with different yeast cell clusters, either number or distribution changes, shows the potential of our 2D light scattering static cytometer for cellular diagnostics.

Lens-free in-line Holographic Microscopy (LHM) is a promising imaging technique for many biomedical and industrial applications. The main advantage of the technique is the simplicity of the imaging hardware, requiring no lenses nor high-precision mechanical components. Nevertheless, the LHM systems achieve high imaging performance only in combination with a high-quality and complex illumination. Furthermore, to achieve truly high-throughput imaging capabilities, many applications require a complete on-chip integration. We demonstrate the strength, versatility and scalability of our integrated approach on two microscopes-on-chip instances that combine image sensor technologies with photonics (and micro-fluidics): a fully integrated Point-Source (PS) LHM module for in-flow cell inspection and Large Field-of-View (LFoV) microscope with on-chip photonic illumination for large-area imaging applications. The proposed PS-LHM module consists of a photonic illumination, a micro-fluidic channel and an imager, integrated in a total volume smaller than 0.5 mm³. A low-loss single-mode photonic waveguide is adapted to generate a high- NA illumination spot. Experimental results show strong focusing capabilities and sufficient overall coupling efficiency. Current PS-LHM prototype reaches imaging resolution below 600nm. Our LFoV-LHM system is extremely vertically compact as it consists of only one 1mm-thick illumination chip and one 3mm-thick imaging module. The illumination chip is based on fractal-layout phase-matched waveguides designed to generate multiple light sources that create a quasi-planar illumination wavefront over an area few square millimeter large. Current illumination prototype has active area of approximately 1.2×1.2mm². Our LFoV-LHM prototype reaches imaging resolution of 870nm using image sensor with 1.12μm pixel pitch with maximum FoV of 16.47mm².

The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells is continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. Limbal Stem Cell Deficiency (LSCD), in which the stem cell population is depleted, can lead to blindness. LSCD can be caused by chemical and thermal burns to the eye. A popular treatment, especially in emerging economies such as India, is the transplantation of limbal stem cells onto damaged limbus with hope of repopulating the region. Hence regenerating the corneal epithelium. In order to gain insights into the success rates of this treatment, new imaging technologies are needed in order to track the transplanted cells.

Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images of the retina. A custom OCT system has been built to image the corneal surface, to investigate the fate of transplanted limbal stem cells. We evaluate two methods to label and track transplanted cells: melanin labelling and magneto-labelling. To evaluate melanin labelling, stem cells are loaded with melanin and then transplanted onto a rabbit cornea denuded of its epithelium. The melanin displays strongly enhanced backscatter relative to normal cells. To evaluate magneto-labelling the stem cells are loaded with magnetic nanoparticles (20-30nm in size) and then imaged with a custom-built, magneto-motive OCT system.

There is a need for development of non-invasive technique to evaluate regenerative tissues such as cell sheets for transplantation. We demonstrated non-invasive imaging inside living cell sheets of human oral mucosal epithelial cells by phase-diversity homodyne optical coherence tomography (OCT). The new method OCT developed in Hitachi enables cell imaging because of high resolution (axial resolution; ~2.6 μm, lateral resolution; ~1 μm, in the air). Nuclei inside cell sheets were imaged with sufficient spatial resolution to identify each cell. It suggested that the new method OCT could be useful for non-invasive cell sheet evaluation test.

High-throughput cell sorting with flow cytometers is an important tool in modern clinical cell studies. Most cytometers use biomarkers that selectively bind to the cell, but induce significant changes in morphology and inner cell processes leading sometimes to its death. This makes label-based cell sorting schemes unsuitable for further investigation. We propose a label-free technique that uses a digital inline holographic microscopy for cell imaging and an integrated, optical neural network for high-speed classification. The perspective of dense integration makes it attractive to ultrafast, large-scale cell sorting. Network simulations for a ternary classification task (monocytes/granulocytes/lymphocytes) resulted in 89% accuracy.

We report on our recent results on robust identification of single bacterial cells embedded in various environments using Spontaneous Raman Scattering. Five species of bacteria were considered, two of which (B. Subtilis and E. Coli) were grown under various conditions, or embedded in two real-world matrices. We recorded the Raman spectra of single cells with a confocal instrument developed in our lab, and performed identification at the species level. Our system integrates a Lensfree imaging module that allows fast detection of bacteria over a large Field-Of-View. Identification rates comparable to those obtained on lab cultures were possible using a comprehensive database containing spectra from bacteria in all environments. In addition, B. Subtilis was correctly identified in 95.5% of the cases using a database composed exclusively of spectra obtained in standard conditions. This is very promising for pathogen threat detection where the construction of an exhaustive database may be challenging.

The luminescence lifetime as a beneficial analytical parameter is known for many years and is well described by a large variety of publications. Many instruments including 2D measuring systems with cameras have been developed and applied in the past years. However, since the current instrumentation to perform either time- or frequency-domain lifetime measurements is rather complex, new developments in CMOS image sensor technology have achieved to create new image sensors, which can efficiently be integrated into easier-to-handle luminescence lifetime measuring systems. The principle of these modulatable CMOS image sensors, while initially being designed for distance measurements, shows a clear analogy to frequency-domain FLIM measurements, which was proven by researchers [1, 2]. Based on this principle a new CMOS image sensor has been developed, integrated into a camera system and has been investigated within a research project. The image sensor has a resolution of 1024 × 1024 pixels with a 5.6 μm pitch and can be modulated up to 50 MHz. First measurements show an effective dynamic range of larger than 1:1024 (corresponding to 10 bit dynamic). The maximum frame rate is in the range of 90 frames/s in dual-tap mode, resulting in an effective lifetime image frame rate for realistic measurements of approximately 22 frames/s. The camera system pco.flim, featuring that image sensor, generates all required modulation signals from 5 kHz to 50 MHz (sinusoidal and rectangular). It performs advanced pixel correction to generate linear and high-quality images, while the basic lifetime image processing is done in the computer. The modulation frequency can be freely adjusted within the specified range. The characteristics of the camera systems are presented, and first results are discussed using different representations of the data like for example the phasor approach [3], which has been established to provide a more global view to pixelwise fluorescence lifetime data and compare time- and frequency-domain results. Based on these results and the experiences of the on-going tests, it can be expected, that the pco.flim will significantly ease the introduction of luminescence lifetime imaging systems to broader applications.

Optical probes to identify tumor margins in vivo would greatly reduce the time, effort and complexity in the surgical removal of malignant tissue in head and neck cancers. Current approaches involve visual microscopy of stained tissue samples to determine cancer margins, which results in the excision of excess of tissue to assure complete removal of the cancer. Such surgical procedures and follow-on chemotherapy can adversely affect the patient’s recovery and subsequent quality of life. In order to reduce the complexity of the process and minimize adverse effects on the patient, we investigate ex vivo tissue samples (stained and unstained) using digital holographic microscopy in conjunction with spectroscopic analyses (reflectance and transmission spectroscopy) in order to determine label-free, optically identifiable characteristic features that may ultimately be used for in vivo processing of cancerous tissues. The tissue samples studied were squamous cell carcinomas and associated controls from patients of varying age, gender and race. Holographic microscopic imaging scans across both cancerous and non-cancerous tissue samples yielded amplitude and phase reconstructions that were correlated with spectral signatures. Though the holographic reconstructions and measured spectra indicate variations even among the same class of tissue, preliminary results indicate the existence of some discriminating features. Further analyses are presently underway to further this work and extract additional information from the imaging and spectral data that may prove useful for in vivo surgical identification.

Here, we present a serial OCT/confocal scanner for histological study of the mouse brain. Three axis linear stages combined with a sectioning vibratome allows to cut thru the entire biological tissue and to image every section at a microscopic resolution. After acquisition, each OCT volume and confocal image is re-stitched with adjacent acquisitions to obtain a reconstructed, digital volume of the imaged tissue. This imaging platform was used to investigate correlations between white matter and microvasculature changes in aging mice. Three age groups were used in this study (4, 12, 24 months). At sacrifice, mice were transcardially perfused with a FITC containing gel. The dual imaging capability of the system allowed to reveal different contrast information: OCT imaging reveals changes in refractive indices giving contrast between white and grey matter in the mouse brain, while transcardial perfusion of a FITC shows microsvasculature in the brain with confocal imaging.

The fiber-coupled microscope (FCM) enables in vivo imaging at deep sites in the tissues or organs that other optical techniques are unable to reach. To develop FCM-based intravital imaging, we employed a plastic optical fiber (POF) bundle that included more than 10,000-units of polystyrene core and polymethyl methacrylate cladding. Each POF had a diameter of less than 5 μm; the tip of the bundle was less than 0.5 mm wide, and the flexible wire had a length of 1,000 mm. The optical performance of the plastic FCM was sufficient for detection of significant signal changes in an acinus of rat pancreas labeled with a calcium ion–sensitive fluorescent dye. In the future, the potential power of plastic FCM is expected to increase, enabling analysis of structure and organization of specific functions in live cells within vulnerable organs.

We utilized terahertz time-domain spectroscopy (THz-TDS) to investigate the complex dielectric properties of solid polycrystalline material of anhydrous glucose (D-(+)-glucose with purity >99.9%). THz transmission spectra of samples were measured from 0.2 to 2.2 THz. The samples were prepared into tablets with thicknesses of 0.362, 0.447, 0.504, 0.522 and 0.626 mm, respectively. The imaginary part of the complex dielectric function of polycrystalline glucose showed that there were multiple characteristic absorption peaks at 1.232, 1.445, 1.522, 1.608, 1.811 and 1.987 THz, respectively. Moreover, for a given characteristic absorption peak, the real part of the complex dielectric function showed anomalous dispersion within the full width half maximum (FWHM) of the absorption peak. Both finite difference time-domain (FDTD) numerical simulations and experimental results showed that the complex dielectric function of anhydrous polycrystalline glucose fits well with the Lorentz dielectric mode. The plasma oscillation frequency was below the frequency of the light waves suggesting that the light waves passed through the polycrystalline glucose tablets. Calculations based on density functional theory (DFT) showed that the characteristic absorption peaks of polycrystalline glucose originated mainly from collective intermolecular vibrations such as hydrogen bonds and crystal phonon modes. The THz radiation can excite the vibrational or rotational energy levels of the biological macromolecules. This leads to changes in their spatial configuration or interactions. This study showed that THz-TDS has potential applications in biological and pharmaceutical research and food industry.

The importance of fluorescence lifetime measurement as an optical analysis tool is growing. Many applications already exist in order to determine the fluorescence lifetime, but the majority of these require the addition of fluorescence-active substances to enable measurements. Every usage of such foreign materials has an associated risk. This paper investigates the use of auto-fluorescing substances in Saccharomyces cerevisiae (Baker’s yeast) as a risk free alternative to fluorescence-active substance enabled measurements. The experimental setup uses a nitrogen laser with a pulse length of 350 ps and a wavelength of 337 nm. The excited sample emits light due to fluorescence of NADH/NADPH and collagen. A fast photodiode collects the light at the output of an appropriate high-pass edge-filter at 400 nm. Fluorescence lifetimes can be determined from the decay of the measurement signals, which in turn characterizes the individual materials and their surrounding environment. Information about the quantity of the fluorescence active substances can also be measured based on the received signal intensity. The correlation between the fluorescence lifetime and the metabolic state of Saccharomyces cerevisiae was investigated and is presented here.

Optical coherence tomography (OCT) provides high resolution, cross-sectional image of internal microstructure of biological tissue. We use the Finite-Difference Time-Domain method (FDTD) to analyze the data acquired by OCT, which can help us reconstruct the refractive index of the biological tissue. We calculate the refractive index tomography and try to match the simulation with the data acquired by OCT. Specifically, we try to reconstruct the structure of melanin, which has complex refractive indices and is the key component of human pigment system. The results indicate that better reconstruction can be achieved for homogenous sample, whereas the reconstruction is degraded for samples with fine structure or with complex interface. Simulation reconstruction shows structures of the Melanin that may be useful for biomedical optics applications.

Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

Background and Objective: Introduction of focused ion beam (FIB) for transmission electron microscopy (TEM) preparation had enhanced the understanding of materials’ interaction at nanoscale. However, this technique generates localized heat that may possibly have some effect on organic/vital structures during preparation of biological tissues. Therefore, the aim of this study was to investigate the effect of milling with Cryogenic-FIB on imaging the ultra-morphological features of dentin-resin interface bonded in a tooth and compare the findings to a room-temperature FIB prepared specimens. Methods: After cylindrical dentin cavities (3 mm diameter × 1.5 mm depth) were prepared on the occlusal surfaces of extracted, non-carious human premolar teeth, they were restored with Filtek P90 (Silorane) restorative system (3M ESPE, USA). To investigate the ultra-morphological features of resin-dentin interface, the bonded specimens were divided into 2 groups based on the preparation technique; (1) FIB preparation at room-temperature (RT), and (2) FIB preparation with cryogenic cooling (Cy). Later, each group was examined under TEM. Results: The obtained sections in RT group showed blurred scattered needle-like crystals above the resin-impregnated dentin. However, the orientation of these crisscross needle-like crystals and the ultramorphological features of the underlying dentin were more vivid and distinct in Cy group. Conclusion: Within the limitation of this in-vitro study, it could be concluded that combining FIB with cryogenic cooling had preserved the biological organic features of dentin due to minimized beam damage. The presented cryogenic technique should be considered in future FIB/TEM studies involving biological substrates. This research was supported by King Abdulaziz University.