This PDF file contains the front matter associated with SPIE Proceedings Volume 8225 including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Chlorophyll fluorescence spectroscopy is employed to detect and study the time evolution of metal stress of Zea mays L. seedlings aiming polluted soil phytoremediation. The chlorophyll fluorescence spectra of intact leaves are analyzed using 405 nm LED excitation. Red (Fr) and far-red (FFr) emissions around 685 nm and 735 nm, respectively, are examined as a function of the heavy metal concentration. The fluorescence ratio Fr/FFr was employed to monitor the effect of heavy metal upon the physiological state of the plants before signs of visual stress became apparent. The chlorophyll fluorescence analysis permitted detection and evaluation of the damage caused by heavy metal soil contamination in the early stages of the plants growing process, which is not feasible using conventional in vitro spectral analysis.

Surface topography and its dynamic fluctuations in live cultured cells were obtained by low-coherent quantitative phase microscopy (LC-QPM), using a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low coherency of the light-source, only the light reflected at a specific sectioning height of the sample generates interference fringes on the CCD camera. Because the digital holographic technique enables us to quantitatively measure the intensity and phase of the optical field, a nanometer-scale surface profile of a living cell can be obtained by capturing the light reflected by the cell membrane. The lateral and the vertical spatial resolution was 0.56 microns and 0.93 microns, respectively, and the mechanical stability of the phase measurement was better than 2 nanometers. The measurements were made at fast (21 frames/sec) and slow (2 frames/sec, time-lapse) frame rates and the slow measurements were performed over a period of 10 minutes. The temporal fluctuations of the cell membrane were analyzed by the mean-square-displacement (MSD) as a function of the time-difference τ. By merging the fast and slow data, the MSDs from τ = 50 msec to τ = 300 sec were obtained and wide-dynamic-range measurements of the MSDs from 2 nm² to over 100000 nm² were demonstrated. The results show significant differences among different cell types under various conditions.

Due to easy integration with microfluidic devices, fiber optical trapping is finding increasing use to immobilize microscopic objects for physical and spectroscopic analysis of samples in solution. Though conventional microscope objective based laser spanners (based on transfer of spin or orbital angular momentum) are being employed for rotational manipulation of microscopic objects, the working distance inside microfluidic channel is limited in depth. Recently, we have demonstrated fiber optic tweezers for in-depth trapping of microscopic objects. Here, we demonstrate the development of a fiber optic spanner for rotation of microscopic objects. This single-mode fiber optics based method does not require special structure or optical properties of the sample to be rotated. Trapping and simultaneous rotation of microscopic objects around axis perpendicular to optic axis could be achieved. The rotation speed of the trapped object could be changed by adjusting the laser beam power coupled to the fiber optic trap. Since the fiber optic spanner could be easily integrated onto a microfluidic platform, the proposed configuration can find potential applications in lab-on-achip devices and tomographic imaging.

Optical trapping of single biological cells has become an established technique for controlling and studying fundamental behavior of single cells with their environment without having "many-body" interference. The development of such an instrument for optical diagnostics (including Raman and fluorescence for molecular diagnostics) via laser spectroscopy with either the "trapping" beam or secondary beams is still in progress. This paper shows the development of modular multi-spectral imaging optical tweezers combining Raman and Fluorescence diagnostics of biological cells.

Embryonal carcinoma (EC) cells, which are cell lines derived from teratocarcinomas, have characteristics in common with stem cells and differentiate into many kinds of functional cells. Similar to embryonic stem (ES) cells, undifferentiated EC cells form multi-layered spheroids. In order to visualize the three-dimensional structure of multilayered EC cells without labeling, we employed full-field interference microscopy with the aid of a low-coherence quantitative phase microscope, which is a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low-coherency of the light-source (halogen lamp), only the light reflected from reflective surface at a specific sectioning height generates an interference image on the CCD camera. P19CL6 EC cells, derived from mouse teratocarcinomas, formed spheroids that are about 50 to 200 micrometers in diameter. Since the height of each cell is around 10 micrometers, it is assumed that each spheroid has 5 to 20 cell layers. The P19CL6 spheroids were imaged in an upright configuration and the horizontally sectioned reflection images of the sample were obtained by sequentially and vertically scanning the zero-path-length height. Our results show the threedimensional structure of the spheroids, in which plasma and nuclear membranes were distinguishably imaged. The results imply that our technique is further capable of imaging induced pluripotent stem (iPS) cells for the assessment of cell properties including their pluripotency.

Fluorescence redox imaging was employed to monitor the metabolic activity of primary human oral keratinocytes prior to the development of tissue-engineered constructs. Keratinocytes with controlled culture conditions were treated with varying levels of chemical stimuli, resulting in differing cellular morphology, growth rate, and metabolic activity. Fluorescence images of keratinocytes were noninvasively acquired from endogenous intracellular metabolic fluorophores NAD(P)H and FAD. A redox ratio quantitatively analyzed each pair of images, showing that fluorescence redox imaging may be a novel technique to characterize live cell viability

The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

The ability to quantitatively and non-invasively detect nanoparticles in tissues has important implications on their development as an in-vivo cancer diagnostic tool. The Diffusion Reflection (DR) method is a simple, non-invasive imaging technique which has been proven useful for the investigation of tissue's optical parameters. We present a new method for the measurements of the GNR concentration in tissue-like phantoms based on DR measurements and the intense light absorption of gold nanorods (GNR). Monte Carlo simulations and tissue-like phantom measurements of the reflected light intensity are presented. The ability to extract the phantoms' optical properties and the GNR concentration in it from DR measurements was demonstrated.

We have developed a model-based, parallel procedure to estimate fluorescence lifetimes. Multiple frequencies are present in the excitation signal. Modeling the entire fluorescence and measurement process produces an analytical ratio of polynomials in the lifetime variable τ. A non-linear model-fitting procedure is then used to estimate τ. We have analyzed this model-based approach by simulating a 10 μM fluorescein solution (τ = 4 ns) and all relevant noise sources. We have used real LED data to drive the simulation. Using 240 μs of data, we estimate τ = 3.99 ns. Preliminary experiments on real fluorescent images taken from fluorescein solutions (measured τ = 4.1 ns), green plastic test slides (measured τ = 3.0 ns), and GFP in U2OS (osteosarcoma) cells (measured τ = 2.1 ns) demonstrate that this model-based measurement technique works.

We have built an all-solid-state camera which is directly modulated at the pixel level for frequency domain fluorescence lifetime imaging microscopy (FLIM) measurement. This novel camera eliminates the need for an image intensifier through the use of an application-specific CCD design, which is being used in a frequency domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and contrast modulation transfer function have been studied through experiments. We are able to do lifetime measurement using MEM-FLIM cameras for various objects, e.g. fluorescence plastic test slides, fluorescein solution, fixed GFP cells, and GFP - Actin stained live cells.

Isolation of different cell types from mixed samples in one separation step by FACS is feasible but expensive and slow. It is cheaper and faster but still challenging by magnetic separation. An innovative bead-based cascade-system (pluriSelect GmbH, Leipzig, Germany) relies on simultaneous physical separation of different cell types. It is based on antibody-mediated binding of cells to beads of different size and isolation with sieves of different mesh-size. We validated pluriSelect system for single parameter (CD3) and simultaneous separation of CD3 and CD15 cells from EDTA blood-samples. Results were compared with those obtained by MACS (Miltenyi-Biotech) magnetic separation (CD3 separation). pluriSelect separation was done in whole blood, MACS on Ficoll gradient isolated leukocytes, according to the manufacturer's protocols. Isolated and residual cells were immunophenotyped (7-color 8-antibody panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLADR) on a CyFlowML flow cytometer (Partec GmbH). Cell count (Coulter), purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (92-98%), yield (50-60%) and viability (92-98%) of isolated cells. PluriSelect separation was slightly faster than MACS (1.15 h versus 1.5h). Moreover, no preenrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two cell subpopulation directly from whole blood and can provide a simple alternative to FACS. The isolated cells can be used for further research applications.

Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

Time-gated luminescence images were obtained by analog summation of a series of sequential images that were obtained with a cooled modified interline CCD camera, and a fluorescence microscope modified to use a UV LED for illumination. The interline CCD camera obtains an analog sum of a multi-frame image by not reading out the storage line after each frame is acquired; instead, the charges from the acquisition pixels are transferred to the storage pixels, which adds them to those previously stored; subsequently, the sum of the images is readout from the storage pixels and digitized. The length of the exposure is limited by the capacity of the storage pixels and the rate of generation of background (noise). Previously, the quality of the images obtained with the room temperature camera was degraded by the buildup of thermal noise. The interline transfer, electronically shuttered, cooled astronomy CCD camera, which was modified for analog summation rapidly produced low noise images; yet permitted long exposures. The past problems with lanthanide dyes of low extinction coefficients and equipment cost have now been solved.

Colony counting is a procedure used in microbiology laboratories for food quality monitoring, environmental management, etc. Its purpose is to detect the level of contamination due to the presence and growth of bacteria, yeasts and molds in a given product. Current automated counters require a tedious training and setup procedure per product and bacteria type and do not cope well with diversity. This contrasts with the setting at microbiology laboratories, where a wide variety of food and bacteria types have to be screened on a daily basis. To overcome the limitations of current systems, we propose the use of hyperspectral imaging technology and examine the spectral variations induced by factors such as illumination, bacteria type, food source and age and type of the agar. To this end, we perform experiments making use of two alternative hyperspectral processing pipelines and compare our classification results to those yielded by color imagery. Our results show that colony counting may be automated through the automatic recovery of the illuminant power spectrum and reflectance. This is consistent with the notion that the recovery of the illuminant should minimize the variations in the spectra due to reflections, shadows and other photometric artifacts. We also illustrate how, with the reflectance at hand, the colonies can be counted making use of classical segmentation and classification algorithms.

Fourier domain optical coherence tomography (FD-OCT) can acquire cross-sectional images of scattering media. Reconstruction of FD-OCT images requires taking the Fourier Transform of the raw data; the accuracy of which depends on the processing algorithms used, among other things. To use the Fast Fourier Transform, data must be evenly sampled in wavenumber, which they generally are not. Thus, researchers must apply hardware- or softwarebased methods to resample the data accordingly. To the best of our knowledge there has been no study that compares different choices for gold standard datasets (i.e., different choices for assigning the sampled wavelengths); furthermore there has been no investigation of the effects of these processing steps on phase data. We first compare different options for gold standard datasets and then examine the effects of various resampling techniques on reconstructed OCT data. We find that an alternative wavenumber sampling range may yield more accurate results for OCT phase data and better intensity of reflector peaks.

To date the flow cytometry (FCM) industry is booming with new generations of commercial clinical instruments. Long-term clinical studies have the dilemma that moving to new instruments being capable of more complex cell-analysis makes it difficult to compare new data with those obtained on older instruments with less complex analysis panels. Since 15 years we conduct follow-up studies on children with congenital heart diseases. In this period we moved from 2- to 3- and now to 10-color FCM immunophenotyping panels. Questions arise how to compare and transfer data from lower to higher level of complexity. Two comparable antibody panels for leukocyte immunophenotyping (12-tube 2-colors, and 9-tube 4-colors) were measured on a BD FACScalibur FCM (calibration: Spherotech beads) in 19 blood samples from children with congenital heart disease. This increase of colors was accompanied by moving antibodies that were in the 2-color panel either FITC or PE labeled to red dyes such as PerCP or APC. Algorithms were developed for bridging data for quantitative characterization of antigen expression (mean fluorescence intensity) and frequency of different cell subpopulations in combination with rainbow bead standard data. This approach worked for the most relevant antibodies (CD3, CD4, CD8 etc.) well, but rendered substantial uncertainty for activation markers (CD69 etc.). Our techniques are particularly well suited to the analysis in long-term studies and have the potential to compare older and recent results in a standardized way.

F_oF₁-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal protondriven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP). According to the 'chemiosmotic hypothesis', a series of proton pumps generate the necessary pH difference plus an electric potential across the bacterial plasma membrane. These proton pumps are redox-coupled membrane enzymes which are possibly organized in supercomplexes, as shown for the related enzymes in the mitochondrial inner membrane. We report diffusion measurements of single fluorescent F_oF₁-ATP synthases in living E. coli by localization microscopy and single enzyme tracking to distinguish a monomeric enzyme from a supercomplex-associated form in the bacterial membrane. For quantitative mean square displacement (MSD) analysis, the limited size of the observation area in the membrane with a significant membrane curvature had to be considered. The E. coli cells had a diameter of about 500 nm and a length of about 2 to 3 μm. Because the surface coordinate system yielded different localization precision, we applied a sliding observation window approach to obtain the diffusion coefficient D = 0.072 μm²/s of F_oF₁-ATP synthase in living E. coli cells.

Bioluminescence imaging (BLI) is an optical molecular imaging modality for monitoring physiological and pathological activities at the molecular level. The information of bioluminescent probe distribution in small animals can be threedimensionally and quantitatively obtained by bioluminescence tomography (BLT). Due to ill-posed nature, BLT may bear multiple solutions and aberrant reconstruction in the presence of measurement noise and optical parameter mismatches. Among different regularization methods, L2-type regularization strategy is the most popular and commonly-applied method, which minimizes the output-least-square formulation incorporated with the l2-norm regularization term to stabilize the problem. However, it often imposes over-smoothing on the reconstruction results. In contrast, for many practical applications, such as early detection of tumors, the volumes of the bioluminescent sources are very small compared with the whole body. In this paper, L1 regularization is used to fully take advantage of the sparsity prior knowledge and improve both efficiency and stability. And then a reconstruction method based on the augmented Lagrangian approach is proposed, which considers the BLT problem as the constrained optimization problem and employs the Bregman iterative method to deal with it. By using "divide and conquer" approach, the optimization problem can be exactly and fast solved by iteratively solving a sequence of unconstrained subproblems. To evaluate the performance of the proposed method in turbid mouse geometry, stimulate experiments with a heterogeneous 3D mouse atlas are conducted. In addition, physical experiments further demonstrate the potential of the proposed algorithm in practical applications.

Role of small molecules such as drugs or metabolites in cells is commonly studied by fluorescence microscopy in which a fluorescent label is attached to the molecule. However, fluorescent labels are typically large that often interfere with the normal cellular function of the molecule. To avoid the use of bulky fluorescent labels, we introduce a technique that uses a simple small chemical tag called alkyne consisting of two carbons connected by a triple bond. The alkyne-tagged molecule is imaged using Raman microscopy that detects the strong Raman signal from the CC triple bond stretching vibration (~2120 cm^-1). Because the alkyne signal is located in the silent region of the cell (1800-2700 cm^-1), it does not interfere with any intrinsic cellular Raman signals. Here, we demonstrate this technique by showing Raman images of an alkyne-tagged component of DNA in a living cell using a slit-scanning confocal Raman microscope. This fast imaging technique is based on a line-shaped focus illumination and simultaneous detection of the Raman spectra from multiple points of the sample. Using this microscope, we obtained time-course Raman images of the incorporation of EdU in the DNA of HeLa cells in just several tens of minutes.

We report on a new two-photon cryomicroscope which consist of a compact laser-scanning microscope combined with a motorized heating and freezing stage. Samples can be cooled down to -196 °C (77 K) and heated up to 600 °C (873 K) with adjustable heating/freezing rates between 0.01 K / min and 150 K / min. Two-photon imaging is realized by near infrared femtosecond-laser pulse excitation. The abilities of the two-photon cryomicroscope are illustrated in several measurements: imaging of fluorescent microspheres inside a piece of ice illustrates the feasibility of deep-microscopic imaging inside frozen sample. The temperature-dependent structural integrity of collagen is monitored by detection of second harmonic generation signals from porcine cornea. The measurements reveal also the dependence of the collagendenaturation temperature on hydration state of the cornea collagen. Furthermore, the potential of the two-photon cryomicroscope for optimization of freezing and thawing procedures as well as to evaluate the viability of frozen cells and tissue is discussed.

Fluorescence spectroscopy has been demonstrated as a viable tool for noting subtle biochemical changes that occur during early-stage cervical cancer progression. Due to multiple fluorophore contributions, the individual fluorophore activities often get masked due to overlapping spectra of neighboring fluorophores. Recently synchronous fluorescence spectroscopy has been demonstrated as an efficient technique for investigation of such non-dominant fluorophores. With synchronous fluorescence spectroscopy individual fluorophore responses are highlighted as sharp peaks by choosing appropriate offsets during signal acquisition. Such peaks may, however be missed due to absorption effects. By correcting the measured synchronous fluorescence spectrum with elastic scattering data, it was observed that the masked fluorophores are highlighted while the broader bands are sharpened. Interestingly, fluorophore activities of protoporphyrin, collagen, NADH, FAD and porphyrin can now be studied using this technique, as compared to only collagen and NADH seen earlier. The results have been verified using tissue phantoms with known fluorophores and scatterers. Use of normalized synchronous spectra has led to enhancement of several fluorophore responses. It was also observed that among the different offsets, the lower ones show sharper features, whereas the larger offsets show a broadband response. Among the different offsets 120nm is found optimal for further investigation.

Enzyme dynamics control and contribute to myriads of mostly well-characterized metabolic processes. Yet, understanding the role of the aqueous reaction matrix remains an experimental challenge. By kinetic THz absorption (KITA) spectroscopy, we have studied the dynamic interplay between water and a human metalloenzyme at work in realtime. In our KITA setup, we combined a THz-time domain spectrometer (THz-TDS) with a stopped-flow mixer to study reactions with millisecond time resolution. We used picosecond THz pulses which directly probe hydrogen bond formation and breaking in the water network to observe enzyme-water interactions upon enzyme catalysis at the active site of a matrix-metalloprotease. During formation of the productive Michaelis complex, we detected a perturbation of coupled enzyme-water network dynamics. Supplemented by real-time biophysical techniques and molecular dynamics simulations we characterized the enzyme-water interplay in the particular case of enzyme catalysis. Our results suggest a polarization-induced gradient of water dynamics at the remote active site of a metalloenzyme with decelerated hydration water dynamics towards the active site. The observed long-range gradient of collective water motions might facilitate productive binding of substrates to enzyme active sites. Further KITA experiments shall improve our understanding of water's contribution to biological function.

Clinical skin-lesion diagnosis uses dermoscopy: 10X epiluminescence microscopy. Skin appearance ranges from black to white with shades of blue, red, gray and orange. Color is an important diagnostic criteria for diseases including melanoma. Melanin and blood content and distribution impact the diffuse spectral remittance (300-1000nm). Skin layers: immersion medium, stratum corneum, spinous epidermis, basal epidermis and dermis as well as laterally asymmetric features (eg. melanocytic invasion) were modeled in an inhomogeneous Monte Carlo model.

Keratins are one of most widely used markers for oral cancers. Keratin 8 and 18 are expressed in simple epithelia and perform both mechanical and regulatory functions. Their expression are not seen in normal oral tissues but are often expressed in oral squamous cell carcinoma. Aberrant expression of keratins 8 and 18 is most common change in human oral cancer. Optical-spectroscopic methods are sensitive to biochemical changes and being projected as novel diagnostic tools for cancer diagnosis. Aim of this study was to evaluate potentials of Raman spectroscopy in detecting minor changes associated with differential level of keratin expression in tongue-cancer-derived AW13516 cells. Knockdown clones for K8 were generated and synchronized by growing under serum-free conditions. Cell pellets of three independent experiments in duplicate were used for recording Raman spectra with fiberoptic-probe coupled HE-785 Raman-instrument. A total of 123 and 96 spectra from knockdown clones and vector controls respectively in 1200-1800 cm^-1 region were successfully utilized for classification using LDA. Two separate clusters with classification-efficiency of ~95% were obtained. Leave-one-out cross-validation yielded ~63% efficiency. Findings of the study demonstrate the potentials of Raman spectroscopy in detecting even subtle changes such as variations in keratin expression levels. Future studies towards identifying Raman signals from keratin in oral cells can help in precise cancer diagnosis.

In vivo multiphoton-intensity images and emission spectra of human skin are reported. Optical sections from different depths of the epidermis and dermis have been measured with near-infrared laser-pulse excitation. While the intensity images reveal information on the morphology, the spectra show emission characteristics of main endogenous skin fluorophores like keratin, NAD(P)H, melanin, elastin and collagen as well as of second harmonic generation induced by the excitation-light interaction with the dermal collagen network.

Exact detection and complete removal of cancer is a key point to minimize cancer recurrence. However, it is currently very difficult to detect small tumors inside human body and continuously monitor tumors using a non-invasive imaging modality. Presently, positron emission tomography (PET) can provide the most sensitive cancer images in the human body. However, PET imaging has very limited imaging time because they typically use isotopes with short halflives. PET imaging cannot also visualize anatomical information. Magnetic resonance imaging (MRI) can provide highresolution images inside the body but it has a low sensitivity, so MRI contrast agents are necessary to enhance the contrast of tumor. Near infrared fluorescent (NIRF) imaging has a good sensitivity to visualize tumor using optical probes, but it has a very limited tissue penetration depth. Therefore, we developed multi-modality nanoparticles for MRI based diagnosis and NIRF imaging based surgery of cancer. We utilized glycol chitosan of 350 nm as a vehicle for MRI contrast agents and NIRF probes. The glycol chitosan nanoparticles were conjugated with NIRF dye, Cy5.5 and bladder cancer targeting peptides to increase the internalization of cancer. For MR contrast effects, iron oxide based 22 nm nanocubes were physically loaded into the glycol chitosan nanoparticles. The nanoparticles were characterized and evaluated in bladder tumor bearing mice. Our study suggests the potential of our nanoparticles by both MRI and NIRF imaging for tumor diagnosis and real-time NIRF image-guided tumor surgery.

In this work we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus FV300) to include nonlinear optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). The NLO microscopies included two-photon fluorescence (TPFE), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG). The whole system, including FLIM, used only one laser source composed of an 80 MHz femtosecond laser. The commercial Ti:sapphire lasers can be tuned up to 690-1040 nm bringing the THG signal to the 350 nm region where most microscope optics do not work. However, the third harmonic is only generated at the sample, meaning that we only have to take care of the collection optics. To do that we used a remote photomultiplier to acquire the THG signal at the 310-350 nm wavelength window. After performing the tests to guarantee that we are observing actually SHG/THG signals we than used this system to acquire multimodal images of several biological samples, from epithelial cancer to vegetables. The ability to see the collagen network together with the cell nuclei proved to be important for cancer tissues diagnosis. Moreover, FLIM provides information about the cell metabolism, also very important for cancer cell processes.

Optogenetics is the science where recent progresses in the field of photonics are combined with the techniques in molecular genetics to develop a methodology for modulation of neural activities.^1-9 Despite enormous enthusiasm in using optogenetics for brain studies, little has been done on the engineering side such as technology development for light delivery or realization of reliable systems for optical monitoring of the induced activities. In this project, we have implemented a Digital Micromirror Device based microprojection system capable of delivering illumination patterns through a high-resolution imaging fiber bundle that guides the pattern to the region of interest on the surface or within the brain tissue. The system is also equipped with an imaging path for detection of calcium signals and monitoring the induced patterns of cellular activities. A very interesting application of the system is extracting topographic computational maps of cortex or cellular receptive fields in-vivo. It is known that such maps are the engine of information processing in the cortex. Better understanding of the structure of such maps will help to unravel the mysteries of brain higher level computations. Another application of this system is related to the high-resolution stimulation patterns that cannot be produced with electrode arrays. Production of high-resolution patterns is important in the study of specific modes of brain activities. We report the details of our optical design, preliminary results produced by testing the system on tissue, and we discuss our strategy to extract new data from the brain tissue.

LED-induced chlorophyll fluorescence spectral signatures from Saccharum officinarum leaves are employed to evaluate the effect of water deficit upon the plant growing process. The chlorophyll fluorescence spectral analysis is a nondestructive and nonintrusive indicator of the chlorophyll content of leaves and abiotic stress intensity, and is used to monitor the time evolution of the effect of water deficit stress upon plants physiological state. Red and far-red emission signals around 685 nm and 735 nm, respectively, are observed and examined as a function of irrigation amount. The intensity ratio of the fluorescence emissions allow one to detect signs of damage in the early stages of the plants growing process, and before traces of visible stress became apparent. The results indicated an unusual behavior of the fluorescence ratio for water stress that can potentially be used as a discriminator amongst several abiotic stresses.

The degenerative joint disease such as osteoarthritis (OA) is closely associated with the death of chondrocytes in apoptosis fashion. Hydrogen peroxide (H₂O₂), higher expression following acute damage in OA patients, has been shown to be up-regulated during apoptosis in a bulk of experimental models. This study was aimed to explore the mechanism of H2O2-induced rabbit chondrocytes apoptosis. Articular cartilage was biopsied from the joints of 6 weeks old New Zealand rabbits. Cell Counting Kit (CCK-8) assay was used to assess the inhibitory effect of H₂O₂ on cell viability. H₂O₂ treatment induced a remarkable reduction of cell viability. We used flow cytometry to assess the form of cell death with Annexin-V/PI double staining, and found that H2O2 treatment induced apoptosis in a dose-and time-dependent manner. Exposure of chondrocytes to 1.5 mM of H₂O₂ for 2 h induced a burst apoptosis that can be alleviated by N-acetyl cysteine (NAC) pretreatment, an anti-oxidant amino-acid derivative. Loss of mitochondria membrane potential (▵Ψm) was evaluated using confocal microscopy imaging and flow cytometry (FCM). H₂O₂ treatment induced a marked reduction of ▵Ψm, and the abrupt disappearance of ▵Ψm occurred within 5 minutes. These results indicate that H₂O₂ induces a rapid apoptosis via a mitochondrial pathway in rabbit chondrocytes.

Evanescent Waves (EWs) and Förster Resonance Energy Transfer (FRET) concepts combined with Atomic Force Microscopy (AFM) have been used for imaging and sensing. For the proposed EW microscopy system, Quantum Dot (QD) embedded Polystyrene microspheres are mounted on AFM cantilevers. When excited by laser, QDs luminescence couples to the Whispering Gallery Modes (WGMs) in the periphery of the microsphere. The resultant EWs extend on the order of 100 nm from the surface of the microsphere. These EWs decay exponentially and are explored as a tool to excite fluorescent labeled trans-membrane and near-membrane proteins. For the FRET system, QD coated silica microspheres are conjugated with fibronectin and mounted on AFM cantilevers. Moving the sphere down to the surface of the RFP-αv integrin tagged cells, fibronectins on the microsphere surface bind to integrins on the cell surface and FRET is observed between the QDs (donor) and RFP (acceptor). The detected fluorescence for the imaging system and the FRET efficiency for the sensing system are both functions of cell surface protein density. These innovative nanoscale imaging and sensing systems can be used to obtain unique dynamic data from living cells to improve understanding of cell adhesion and mechanobiology in cells.

Exposure of skin tissue to UV radiation has been shown to cause DNA photodamage. If this damaged DNA is allowed to replicate, carcinogenesis may occur. DNA damage is prevented from being passed on to daughter cells by upregulation of the protein p21. p21 halts the cells cycle allowing the cell to undergo apoptosis, or repair its DNA before replication. Previous work suggested that milk phospholipids may possess protective properties against UV damage. In this study, we observed cell morphology, cell apoptosis, and p21 expression in tissue engineered epidermis through the use of Hematoxylin and Eosin staining, confocal microscopy, and western blot respectively. Tissues were divided into four treatment groups including: a control group with no UV and no milk phospholipid treatment, a group exposed to UV alone, a group incubated with milk phospholipids alone, and a group treated with milk phospholipids and UV. All groups were incubated for twenty-four hours after treatment. Tissues were then fixed, processed, and embedded in paraffin. Performing western blots resulted in visible p21 bands for the UV group only, implying that in every other group, p21 expression was lesser. Numbers of apoptotic cells were determined by observing the tissues treated with Hoechst dye under a confocal microscope, and counting the number of apoptotic and total cells to obtain a percentage of apoptotic cells. We found a decrease in apoptotic cells in tissues treated with milk phospholipids and UV compared to tissues exposed to UV alone. Collectively, these results suggest that milk phospholipids protect cell DNA from damage incurred from UV light.

Scenes in real world have dynamic range of radiation that cannot be captured by conventional cameras. High dynamic range imaging is a technique to capture detail images where, in the field of image, intensity variation is extreme. This technique is very useful for biological imaging where the samples have very bright and very dark regions and both parts have useful information. In this article we propose a novel high dynamic range imaging technique based on compressive imaging that uses one single detector instead of camera (array of detectors) to capture an image. Combination of high dynamic range imaging and compressive imaging benefits from imaging with high dynamic range of radiation and advantages of compressive sampling; namely, imaging at regions of optical spectrum where conventional cameras are not readily available and single detectors are available. Additionally, as its name suggests, this technique requires less number of samples (compared to raster scanning). Our experimental results show that high dynamic range compressive imaging system is capable of capturing images with large intensity contrast.

With the advent of optogenetics, all optical control and visualization of the activity of specific cell types is possible. We have developed a fiber optic based probe to control/visualize neuronal activity deep in the brain of awake behaving animals. In this design a thin multimode optical fiber serves as the head of the probe to be inserted into the brain. This fiber is used to deliver excitation/stimulation optical pulses and guide a sample of the emission signal back to a detector. The major trade off in the design of such a system is to decrease the size of the fiber and intensity of input light to minimize physical damage and to avoid photobleaching/phototoxicity but to keep the S/N reasonably high. Here the excitation light, and the associated emission signal, are frequency modulated. Then the output of the detector is passed through a time-lens which compresses the distributed energy of the emission signal and maximizes the instantaneous S/N. By measuring the statistics of the noise, the structure of the time lens can be designed to achieve the global optimum of S/N. Theoretically, the temporal resolution of the system is only limited by the time lens diffraction limit. By adding a second detector, we eliminated the effect of input light fluctuations, imperfection of the optical filters, and back-reflection of the excitation light. We have also designed fibers and micro mechanical assemblies for distributed delivery and detection of light.

The solution of the forward problem in fluorescence molecular imaging is among the most important premises for the successful confrontation of the inverse reconstruction problem. To date, the most typical approach has been the application of the diffusion approximation as the forward model. This model is basically a first order angular approximation for the radiative transfer equation, and thus it presents certain limitations. The scope of this manuscript is to present the dual coupled radiative transfer equation and diffusion approximation model for the solution of the forward problem in fluorescence molecular imaging. The integro-differential equations of its weak formalism were solved via the finite elements method. Algorithmic blocks with cubature rules and analytical solutions of the multiple integrals have been constructed for the solution. Furthermore, specialized mapping matrices have been developed to assembly the finite elements matrix. As a radiative transfer equation based model, the integration over the angular discretization was implemented analytically, while quadrature rules were applied whenever required. Finally, this model was evaluated on numerous virtual phantoms and its relative accuracy, with respect to the radiative transfer equation, was over 95%, when the widely applied diffusion approximation presented almost 85% corresponding relative accuracy for the fluorescence emission.

We present a spectrally resolved confocal imaging approach to qualitatively asses the overall uptake and the penetration depth of fluorescent dyes into biological tissue. We use a confocal microscope with a spectral resolution of 5 nm to measure porcine skin tissue after performing a Franz-Diffusion experiment with a submicron emulsion enriched with the fluorescent dye Nile Red. The evaluation uses linear unmixing of the dye and the tissue autofluorescence spectra. The results are combined with a manual segmentation of the skin's epidermis and dermis layers to assess the penetration behavior additionally to the overall uptake. The diffusion experiments, performed for 3h and 24h, show a 3-fold increased dye uptake in the epidermis and dermis for the 24h samples. As the method is based on spectral information it does not face the problem of superimposed dye and tissue spectra and therefore is more precise compared to intensity based evaluation methods.

Utilizing a custom-built, on-stage incubator-combined, two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) imaging system, we observed new-sarcomere addition in rat neonatal cardiomyocytes during 10 hours of on-stage incubation. This addition occurred at one end of an existing myofibril, the sides of existing myofibrils, and at the interstice of several separated myofibrils; in the cases of the latter two, we observed mature myofibrils acting as templates. We found that during sarcomeric addition, myosin filaments are assembled onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric length during maturation.

Retinopathic injuries are a common symptom of many diseases. However, if detected early, much of the damage caused by these injuries can be prevented, or in some cases reversed. In this study, images of retinas were classified as normal or injured using the vascular cell count, vasculature coverage, and vessel caliber. To model retinal vasculopathies, retinal vasculature from mice with the BCL-2 gene either partially or completely knocked out were compared. The bcl-2 gene is a critical regulator of apoptosis and angiogenesis, and therefore its absence has a significant impact on the number of vascular cells and vasculature complexity. When the aforementioned features were extracted from the images, classification was performed using a majority vote between a linear classifier, k-nearest-neighbors classification, and a support vector machine. This resulted in a classification accuracy of 81% using the "leave one out" error determination method.

An ongoing challenge in the field of cell biology is to how to quantify the size and shape of organelles within cells. Automated image analysis methods often utilize thresholding for segmentation, but the calculated surface of objects depends sensitively on the exact threshold value chosen, and this problem is generally worse at the upper and lower zboundaries because of the anisotropy of the point spread function. We present here a threshold-independent method for extracting the three-dimensional surface of vacuoles in budding yeast whose limiting membranes are labeled with a fluorescent fusion protein. These organelles typically exist as a clustered set of 1-10 sphere-like compartments. Vacuole compartments and center points are identified manually within z-stacks taken using a spinning disk confocal microscope. A set of rays is defined originating from each center point and radiating outwards in random directions. Intensity profiles are calculated at coordinates along these rays, and intensity maxima are taken as the points the rays cross the limiting membrane of the vacuole. These points are then fit with a weighted sum of basis functions to define the surface of the vacuole, and then parameters such as volume and surface area are calculated. This method is able to determine the volume and surface area of spherical beads (0.96 to 2 micron diameter) with less than 10% error, and validation using model convolution methods produce similar results. Thus, this method provides an accurate, automated method for measuring the size and morphology of organelles and can be generalized to measure cells and other objects on biologically relevant length-scales.

Oxidative stress (OS), which increases during diabetes, exacerbates the development and progression of diabetes complications including renal vascular and proximal tubule cell dysfunction. The objective of this study was to investigate the changes in the metabolic state of the tissue in diabetic mice kidneys using fluorescence imaging. Mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH-2 (Flavin Adenine Dinucleotide) are autofluorescent and can be monitored without exogenous labels by optical techniques. The ratio of the fluorescence intensity of these fluorophores, (NADH/FAD), called the NADH redox ratio (RR), is a marker of metabolic state of a tissue. We examined mitochondrial redox states of kidneys from diabetic mice, Akita/+ and its control wild type (WT) for a group of 8- and 12-week-old mice. Average intensity and histogram of maximum projected images of FAD, NADH, and NADH RR were calculated for each kidney. Our results indicated a 17% decrease in the mean NADH RR of the kidney from 8-week-old mice compared with WT mice and, a 30% decrease in the mean NADH RR of kidney from12-week-old mice compared with WT mice. These results indicated an increase in OS in diabetic animals and its progression over time. Thus, NADH RR can be used as a hallmark of OS in diabetic kidney allowing temporal identification of oxidative state.

RBC has been shown to possess shape memory subsequent to shear-induced shape transformation. However, this property of RBC may not be generalized to all kinds of stresses. Here, we report our observation on the action of radiation pressure forces on RBC's shape memory using optical manipulation and quantitative phase microscopy (OMQPM). QPM, based on Mach-Zehnder interferrometry, allowed measurement of dynamic changes of shape of RBC in optical tweezers at different trapping laser powers. In high power near-infrared optical tweezers (>200mW), the RBC was found to deform significantly due to optical forces. Upon removal of the tweezers, hysteresis in recovering its original resting shape was observed. In very high power tweezers or long-term stretching events, shape memory was almost erased. This irreversibility of the deformation may be due to temperature rise or stress-induced phase transformation of lipids in RBC membrane.

Angular Domain Imaging (ADI) is an imaging technique that is capable of generating three dimensional images of attenuating targets embedded in a scattering medium. In ADI, an angular filter is positioned between the sample and the detector to discriminate between quasi-ballistic photons and scattered photons. Quasi-ballistic photons have undergone relatively few forward directed scattering events, and can be used to generate a projection image representative of the imaging target. Scattered photons have undergone many scattering events and contain little information regarding the imaging target, thereby leading to decreased image contrast. Our implementation of ADI utilizes a silicon micro-channel array to reject scattered photons based on the angle at which they exit the sample. The objective of this work was to collect ADI images with a tunable pulsed laser within the visible range. Samples were illuminated at 13 wavelengths between 460 nm and 700 nm. An angular filter array of 80 μm × 80 μm tunnels 2-cm long was used to select the quasiballistic photons. Images were detected with a linear 16-bit linear CCD. The phantom consisted of a 0.7 mm attenuating target submerged in one of four Intralipid^R dilutions (0.15%-0.3%) contained within a 1 cm path length cuvette. Image contrast ranged from 0.02 at 460 nm and 0.3% Intralipid^R to 1 at 680 nm at 0.15% Intralipid^R. For a given scattering level, contrast increased at longer wavelengths. Resolution varied minimally with wavelength. The results suggested that multispectral ADI with a tunable pulsed laser is feasible and may find utility in imaging thin tissue samples in the future.

A novel stigmatic mass microscope using laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch were clearly observed, and the estimated spatial resolution was about 3 μm in the linear mode with a 20-fold ion optical magnification. Tissue sections of a brain and eyes of a mouse stained with crystal violet and methylene blue were observed in the linear mode, and the stigmatic total ion images of crystal violet and methylene blue agreed well with the optical photomicrograph of the same sections. Especially, the fine structure in the cornea tissue was clearly observed with a spatial resolution in the range of micrometers. Although the total measurement time of the stigmatic ion image for the whole-eye section was about 59 minutes using a laser with a 10 Hz repetition rate, the measurement time could be reduced to about 35 s using a laser with a 1 kHz repetition rate and automation of measurements. The stigmatic mass microscope developed in this research should be suitable for high-spatial resolution and high-throughput imaging mass spectrometry for pathology, pharmacokinetics, and so on.

Basal cell carcinoma (BCC) is the most common form of skin cancer. To improve the diagnostic accuracy, additional non-invasive methods of making a preliminary diagnosis have been sought. We have implemented an En-Face optical coherence tomography (OCT) for this study in which the dynamic focus was integrated into it. With the dynamic focus scheme, the coherence gate moves synchronously with the peak of confocal gate determined by the confocal interface optics. The transversal resolution is then conserved throughout the depth range and an enhanced signal is returned from all depths. The Basal Cell Carcinoma specimens were obtained from the eyelid a patient. The specimens under went analysis by DF-OCT imaging. We searched for remarkable features that were visualized by OCT and compared these findings with features presented in the histology slices.

Fast detection of bacterial concentrations is important for the food industry and for healthcare. Early detection of infections and appropriate treatment is essential since, the delay of treatments for bacterial infections tends to be associated with higher mortality rates. In the food industry and in healthcare, standard procedures require the count of colony-forming units in order to quantify bacterial concentrations, however, this method is time consuming and reports require three days to be completed. An alternative is metabolic-colorimetric assays which provide time efficient in vitro bacterial concentrations. A colorimetric assay based on Resazurin was developed as a time kinetic assay (KRA) suitable for bacterial concentration measurements. An optimization was performed by finding excitation and emission wavelengths for fluorescent acquisition. A comparison of two non-related bacteria, foodborne pathogens Escherichia coli and Listeria monocytogenes, was performed in 96 well plates. A metabolic and clonogenic dependence was established for fluorescent kinetic signals.