This PDF file contains the front matter associated with SPIE Proceedings Volume 7568, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

A new form of near-field microscopy is presented using digital holography for quantitative phase imagery and characterization of cell-substrate interfaces. This imaging technique, termed total internal reflection holographic microscopy (TIRHM), utilizes an evanescent wave phase shift from the presence of cellular organisms, membranes, adhesions, and tissue structures on a prism face in order to modulate an object beam wavefront in a digital holographic microscope. Quantitative phase images of live cellular specimens are presented.

As the excited state lifetime of a fluorescent molecule depends on its environment, it is possible to use it as a probe for physico-chemical parameters of the surrounding medium. Whereas this is well known for many solid guest/host systems, only few reports of quantitative, temporal resolved in vivo studies to monitor the nano-environment for a protein-coupled chromophore such as GFP are known from literature. Here we present a novel approach to determine the membrane potential of living (plant) cells based on the fluorescence lifetime (FLT) analysis of membrane-located GFP. By using confocal sample scanning microscopy (CSSM) combined with fluorescence lifetime imaging microscopy, we recently showed that the phytohormone brassinolide (BL) induces cell wall expansion and a decrease in the FLT of the BRI1-GFP in living cells of Arabidopsis thaliana seedlings. BRI1 is the dominant functional receptor for BL in Arabidopsis and locates to the plasma membrane. Although the dependence of the FLT of GFP on its physico-chemical environment such as pH-value, refractive index and pressure has been reported, the observed FLT decrease of BRI1-GFP in response to BL application could not be explained by these parameters. However, our in vivo FLT and CSSM analyses indicate that the BLinduced change in the FLT of BRI1-GFP is caused by hyperpolarisation of the plasma membrane (Em). Thus, our results indicate that BRI1-GFP serves as sensitive and non-invasive probe for recording the Em of the plasma membrane in living plant cells with high spatio-temporal resolution.

Endogenous fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), keratin, and tryptophan, have been used as contrast agents for imaging metabolism and morphology of living cells and tissues. Multilabeling which maps the distribution of different targets is an indispensable technique in many biomedical and biochemical studies. Therefore, two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with in vivo fluorescence labeling techniques such as genetically encoded fluorescent protein could be a powerful tool for imaging living cells and tissues. However, the challenge is that the excitation and emission wavelengths of these endogenous fluorophores and fluorescence labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected Supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores and fluorescent proteins such as NADH, tryptophan, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) were excited in their optimal wavelengths alternately or simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and spectral domains. Cellular organelles such as nucleus, mitochondria, microtubule and Endoplasmic Reticulum (ER), were clearly revealed in the TPEF images.

We present the real time quantitative analysis of Vibrio vulnificus-infected host cells using high stability quantitative phase microscopy (HSQPM). It provides the ability to retrieve the phase or optical path length distribution over the cell from a single interferogram image, which has been measured with nanometer path length sensitivity for long periods of time. We have applied HSQPM to study dynamic cell morphologic changes and to quantify noninvasively cell volumes of rat basophilic leukemia RBL-2H3 cells infected with pathogenic bacteria V. vulnificus strains, wild type (MO6-24/O) and RTX toxin mutant (CMM770). During the process of V. vulnificus wild type infection to RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes and areas were observed in real time using HSQPM. In contrast, the dramatic changes were not detected in RBL-2H3 cells infected with RTX toxin mutant. The results showed the good correlation between HSQPM analysis and biochemical assays such as lactate dehydrogenase (LDH) assay and β-hexosaminidase release assay. We suggest that HSQPM is useful real time quantitative method to study the dynamic process of host cells infected with pathogen in a noninvasive manner.

Gold nanorods have unique optical properties as their two photon absorption cross sections are very high and their spectral positions of extinction bands can be controlled by their aspect ratio only, so that gold nanorods have been considered as agents for cell imaging. Two-photon photoluminescence imaging could be used to detect the cellular gold nanorods with the high power femto-second (fs) infrared laser, but may cause the photothermal effect melting the rods. The 3-D distribution of gold nanorods in living cells also can be measured by confocal reflectance microscopy with a very low laser power, and thus the cell damaging can be avoided. In this work, these two methods were comparatively studied in living rat basophilic leukemia (RBL-2H3) cells.

The pace of biomedical research towards reducing the onset of disease is greatly accelerated when out-of-thebox approaches are used to develop a novel technology and tool. The National Cancer Institute's program for Innovative Molecular Analysis Technologies (IMAT) continues to focus on supporting and stimulating investigator-initiated innovation to develop technologies that have the potential for revolutionizing cancer research and care. Also described in this extended abstract are other funding opportunities from the National Institutes of Health (NIH) that may be of interest to those seeking support for technology development.

Multiphoton optical tomography or intravital tomography (IVT) provides non-invasive optical sectioning of biological specimens, e.g. skin, with subcellular spatial resolution without any need of contrast agents. It can be used to distinguish between normal and diseased tissue due to the differences in morphological appearance. Additional information beyond morphology can be obtained by analyzing the collected fluorescence light spectroscopically and by means of its fluorescence decay time. This is frequently termed spectral fluorescence lifetime imaging (SFLIM) or 5D-intravital tomography (5D-IVT). Spectral and temporal resolution scales with the number of detection increments (i.e. spectral channels and time bins). 5D-IVT enables us to detect new physiological parameters, however accompanied by a decrease in intensity per channel. Moreover, the increase of data requests a higher need of software skills. In this study we investigate and evaluate different technical modes of 5D-IVT with respect to their clinical relevance: (1) a multichannel photomultiplier tube (PMT) array coupled to a diffraction grating, each channel being analyzed by timecorrelated single photon counting (TCSPC), (2) three separate PMTs in spectral separation path using dichroic mirrors, each channel being analyzed by TCSPC and (3) a single PMT TCSPC setup in combination with a high-resolution CCDspectrograph for pointwise microspectroscopy.

Fluorescence imaging is a potential candidate for tissue diagnostics in a wide variety of clinical situations. In order to extract diagnostic information using fluorescence, different approaches may be used. Typically, fluorescence imaging is performed by illuminating the sample at a single excitation wavelength and detecting the emissions at one or more wavelengths. We have built a prototype system for a new fluorescence imaging technique denoted Selective Excitation Light Fluorescence (SELF) Imaging. In this technique, the sample is illuminated with multiple excitation wavelengths, and one or more emitted wavelength images are detected. By using a multitude of illumination wavelengths or a weighted sum of illumination wavelengths, SELF imaging can highlight differences in the excitation spectra of fluorophores in the sample. Some potential advantages of this imaging technique are: detection of multiple labeled objects in microscopy using only a single filter cube, increasing the number of simultaneous labels which can be used on a single slide as labels are separated by their absorption spectra not just their emission spectra, detection of different components of tissue based on different excitation spectra, etc.

Laser induced fluorescence is exploited to evaluate the effect of abiotic stresses upon the evolution and characteristics of in vivo chlorophyll emission spectra of leaves tissues of brazilian biofuel plants species(Saccharum officinarum and Jatropha curcas). The chlorophyll fluorescence spectra of 20 min predarkened intact leaves were studied employing several excitation wavelengths in the UV-VIS spectral region. Red(Fr) and far-red (FFr) chlorophyll fluorescence emission signals around 685 nm and 735 nm, respectively, were analyzed as a function of the stress intensity and the time of illumination(Kautsky effect). The Chl fluorescence ratio Fr/FFr which is a valuable nondestructive indicator of the chlorophyll content of leaves was investigated during a period of time of 30 days. The dependence of the Chl fluorescence ratio Fr/FFr upon the intensity of the abiotic stress(salinity) was examined. The results indicated that the salinity plays a major hole in the chlorophyll concentration of leaves in both plants spieces, with a significant reduction in the chlorophyll content for NaCl concentrations in the 25 - 200 mM range. The laser induced chlorophyll fluorescence analysis allowed detection of damage caused by salinity in the early stages of the plants growing process, and can be used as an early-warning indicator of salinity stress

In optical imaging it is thought that optimum tumor contrast can be achieved with the use of small-labeled molecular tracers that have high affinity to their targets and fast clearance rates from the blood stream and healthy tissues. An example of this is fluorescently tagged EGF to monitor the molecular activity of tumors, such as pancreatic cancer. Extensive fluorescence contrast analysis for fluorescence molecular tomography has been performed on the AsPC-1 pancreas tumor, grown orthotopically in mice; yet, the binding dynamics of the EGF-fluorescent agent in vivo is not completely known. The bulk pancreatic tumor displays 3:1 contrast relative to the normal pancreas at long times after injection; however, even higher levels of fluorescence in the liver, kidney and intestine suggest that molecular specificity for the tumor may be low. Mice were administered a fluorescently labeled EGF agent and were sacrificed at various time points post-injection. To analyze the amount of specific binding at each time point frozen tissue samples were fluorescently imaged, washed with saline to remove the interstitially distributed contrast agent, and then imaged again. This technique demonstrated that approximately ~10% of the molecular target was firmly bound to the cell, while 90% was mobile or unbound. This low binding ratio suggests that the contrast observed is from inherent properties of the tumor (i.e. enhanced permeability and retention effect) and not from specific bound contrast as previously anticipated. The use of EGF contrast agents in MRI-guided fluorescence tomography and the impact of low binding specificity are discussed.

Confocal scanning laser ophthalmoscope (CSLO) has been established to be an important diagnostic tool for retinopathies like age-related macular degeneration, glaucoma and diabetes. Compared to a confocal laser scanning microscope, CSLO is also capable of providing optical sectioning on retina with the aid of a pinhole, but the microscope objective is replaced by the optics of eye. Since optical spectrum is the fingerprint of local chemical composition, it is attractive to incorporate spectral acquisition into CSLO. However, due to the limitation of laser bandwidth and chromatic/geometric aberration, the scanning systems in current CSLO are not compatible with spectral imaging. Here we demonstrate a spectral CSLO by combining a diffraction-limited broadband scanning system and a supercontinuum laser source. Both optical sectioning capability and sub-cellular resolution are demonstrated on zebrafish's retina. To our knowledge, it is also the first time that CSLO is applied onto the study of fish vision. The versatile spectral CSLO system will be useful to retinopathy diagnosis and neuroscience research.

Molecular imaging provides the in vivo characterization of cellular molecular events involved in normal and pathologic processes. With the advent of optical molecular imaging, specific molecules, proteins and genes may be tagged with a luminescent reporter and visualized in small animals. This powerful new tool has pushed in vivo optical imaging to the forefront as it allows for direct determination of drug bio-distribution and uptake kinetics as well as an indicator of biochemical activity and drug efficacy. Although optical imaging encompasses diverse techniques and makes use of various wavelengths of light, a great deal of excitement in molecular research lies in the use of tomographic and fluorescence techniques to image living tissues with near-infrared (NIR) light. Nonionizing, noninvasive near-infrared optical imaging has great potential to become promising alternative for breast cancer detection. Fluorescence spectroscopy studies of human tissue suggest that a variety of lesions show distinct fluorescence spectra compared to those of normal tissue. It has also been shown that exogenous dyes exhibit selective uptake in neoplastic lesions and may offer the best contrast for optical imaging. Use of exogenous agents would provide fluorescent markers, which could serve to detect embedded tumors in the breast. In particular, the ability to monitor the fluorescent yield and lifetime may also enable biochemical specificity if the fluorophore is sensitive to a specific metabolite, such as oxygen. As a first step, we have synthesized and characterized one such NIR fluorescent dye conjugate, which could potentially be used to detect estrogen receptors (ER)[2] . The conjugate was synthesized by ester formation between 17-β estradiol and a hydrophilic derivative of indocyanine green (ICG) cyanine dye, bis-1, 1-(4-sulfobutyl) indotricarbocyanine-5- carboxylic acid, sodium salt. The ester formed was found to have an extra binding ability with the receptor cites as compared to ICG, which was established by the partition coefficient studies. The replacement of the sodium ion in the ester by a larger glucosammonium ion was found to enhance the hydrophilicity and reduce the toxic effect on the cell lines. The excitation and emission peaks for the conjugate were recorded in the NIR region as 750nm and 788nm respectively. The ester was found nontoxic on adenocarcinoma breast cancer cell lines MCF-7/MDA-MB-231. Specific binding and endocytosis of the estrogen-labeled conjugate was studied on the MCF-7 (ER positive) and MDA-MB-231 (ER negative). Conjugate staining of MCF-7 cells showed ~ 4-fold increase in signal intensity compared to MDA-MB- 231. Further, estrogen molecules were found to be specifically localized to the nuclear region of MCF-7 cells, whereas MDA-MB-231 showed plasma membrane staining. This technique offers the potential of noninvasive detection of hormone receptor status in breast cancer cells and would help in decreasing the load of unnecessary biopsies. Here, we have reported the progress made in the development of a novel NIR external contrast agent and the work is in progress to use this conjugate for the molecular based, diagnostic imaging of breast cancer.

Elastic fibers are essential components of the human aorta, and there is an association between elastin fibers remodeling and several diseases. Hypertension is one such example of a disease leading to elastin fibers remodeling. These fibers can be easily seen in eosin-hematoxilin (HE) stained histologic sections when observed by UV-excited fluorescence microscopy or by a much more precise Laser Scanning Confocal Microscope (LSCM). In order to study the effect of the hypertension on the elastin fibers pattern we developed an automatic system (software and hardware) to count the number of elastin fibers and to measure the distance between them in a LSCM and used it compare the statistical distribution of the distance between these fibers in normotensive and hypertensive patients. The full image of the whole sample (2 or 3mm long) was composed by several 220×220μm frames with 512×512 pixels. The software counters fiber and distance between fibers. We compared the elastic fiber texture in routinely HE-stained histologic slides of the aorta ascendens in 24 normotensive and 30 hypertensive adult patients of both sexes and of similar age from our autopsy files. Our results show that the average number of fibers is the same for both cases but the distance between the fibers are larger for hypertensive patients than for normotensive ones.

Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample giving insight into the vibration eigenmodes of the excited molecules. Raman spectroscopy provides a detailed chemical composition of the sample, constituting a sort of its chemical fingerprint. Although Raman spectroscopy is a useful technique to identify and quantify species in a given matrix, it has been severely limited in its applicability by fluorescence. Spectrally, this fluorescence occurs at the same wavelength as the Raman signal and is often several orders of magnitude more intense that the weak chemical transitions probed by Raman spectroscopy. Often, this fluorescence background and its natural variability make biochemical analysis using Raman spectroscopy impractical. In this work, we present the theory and the implementation of an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background by modulating of the excitation wavelength. The method is based on the continuous wavelength shift of the Raman peaks with the modulation of the laser wavelength while the fluorescence background remains static. Exploiting this physical property allows us to clearly distinguish between the Raman signal and the fluorescence background. Our method is related to wavelength shifting Raman spectroscopy but incorporates two key novel elements: (i) the use of more than two excitation wavelengths and (ii) multi-channel lock-in detection of the Raman signal for suppression of the fluorescence background. Our results establish a direct and practical approach for fluorescence background suppression in 'real-time' Raman spectroscopy for in-vivo biomedical applications.

Therapeutic treatment of spinal cord injuries, brain trauma, stroke, and neurodegenerative diseases will greatly benefit from the discovery of compounds that enhance neuronal regeneration following injury. We previously demonstrated the use of femtosecond laser microsurgery to induce precise and reproducible neural injury in C. elegans, and have developed microfluidic on-chip technologies that allow automated and rapid manipulation, orientation, and non-invasive immobilization of animals for sub-cellular resolution two-photon imaging and femtosecond-laser nanosurgery. These technologies include microfluidic whole-animal sorters, as well as integrated chips containing multiple addressable incubation chambers for exposure of individual animals to compounds and sub-cellular time-lapse imaging of hundreds of animals on a single chip. Our technologies can be used for a variety of highly sophisticated in vivo high-throughput compound and genetic screens, and we performed the first in vivo screen in C. elegans for compounds enhancing neuronal regrowth following femtosecond microsurgery. The compounds identified interact with a wide variety of cellular targets, such as cytoskeletal components, vesicle trafficking, and protein kinases that enhance neuronal regeneration.

We have shown the potential of a new method for optimizing the separation of human stem cell subsets from peripheral blood based on a novel cell labeling technique that leverages the capabilities of a new commercially available high speed magnetic cell sorting system (IKOTECH LLC, New Albany, IN). This new system sorts cells in a continuously flowing manner using a Quadrupole Magnetic cell Sorter (QMS). The sorting mechanism is based upon the magnetophoretic mobility of the cells, a property related to the relative binding distributions of magnetic particles per cell, as determined by the utilization of a Magnetic Cell Tracking Velocimeter (MCTV). KG-1 cells were competitively labeled with anti-CD34 magnetic beads and anti-CD34 FITC to obtain an optimal level of magnetophoretic mobility as visualized by the MCTV for high throughput sort recovery in the QMS. In QMS sorting, the concept of split-flow thin channel (SPLITT) separation technology is applied by having a sample stream enter a vertical annular flow channel near the channel's interior wall followed by another sheath flow entering near the exterior wall. The two flows are initially separated by a flow splitter. They pass through the bore of a Halbach permanent quadrupole magnet assembly, which draws magnetized cells outward and deflects them into a positive outflow, while negative cells continue straight out via the inner flow lamina. QMS sorts cells based upon their magnetophoretic mobility, or the velocity of a cell per unit ponderomotive force, the counterpart of fluorescence intensity in flow cytometry. The magnetophoretic mobility distribution of a cell population, measured by automated MCTV, is used as input data for the algorithmic control of sample, sheath, and outlet flow velocities of the QMS. In this study, the relative binding distributions of magnetic particles per cell were determined by MCTV using novel sorting and sizing algorithms. The resulting mobility histograms were used to set the QMS flow parameters so that desired cell populations could be selected on the basis of a mobility "window". The MCTV and the QMS are able to work together to provide good sort boundaries for cell populations that are mathematically defined as opposed to the traditional magnetic sort systems that solely rely on whether a cell is simply "magnetized" or not. One long-term application of this new high speed cell sorting system is to sterilely isolate large numbers of human stem cells directly from a donor's blood for subsequent manipulation in tissue culture for regenerative medicine within that same patient. This will eliminate the need for immune suppressive drugs in an autologous transplantation procedure.

Metastatic cells have the ability to break through the basal lamina, enter the blood vessels, circulate through the vasculature, exit at distant sites, and form secondary tumors. This multi-step process, therefore, clearly indicates the inherent ability of metastatic cells to sense, process, and adapt to the mechanical forces in different surrounding environments. We describe a magnetic probing device that is useful in characterizing the mechanical properties of cells along arbitrary two-dimensional directions. Magnetic force, with the advantages of biocompatibility and specificity, was produced by magnetic poles placed in an octupole configuration and applied to fibronectin-coated magnetic microbeads attached on cell membrane. Cell deformation in response to the applied force was then recorded through the displacement of the microbeads. The motion of the beads was measured by computer processing the video images acquired by a high-speed CMOS camera. Rotating force vectors with constant magnitude while pointing to directions of all 360 degrees were applied to study the mechanical anisotropy of metastatic breast cancer cells MDA-MB-231. The temporal changes in magnitude and directionality of the cellular responses were then analyzed to investigate the cellular adaptation to force stimulation. This probing technology thus has the potential to provide us a better understanding of the mechano-signatures of cells.

Atmospheric pressure mass analysis of solid phase biomolecules is performed using laser electrospray mass spectrometry (LEMS). A non-resonant femtosecond duration laser pulse vaporizes native samples at atmospheric pressure for subsequent electrospray ionization and transfer into a mass spectrometer. LEMS was used to detect a complex molecule (irinotecan HCl), a complex mixture (cold medicine formulation with active ingredients: acetaminophen, dextromethorphan HBr and doxylamine succinate), and a biological building block (deoxyguanosine) deposited on steel surfaces without a matrix molecule.

We present MICAO, an accessory which implements adaptive optics for microscopy. The device is designed to be plugged into the camera port of an inverted microscope to correct for the wavefront aberrations introduced by the system. It is compatible with most of the microscopes and can be easily installed in an inverted microscope imaging system. The design of the system allows working with almost all of the biological objectives. We also present the sensorless correction algorithm used to improve the image quality. Finally, we demonstrate the capability of the system to correct the aberrations present into a sample designed specially.

Microtubules are a crucial part of the fungal cytoskeleton and facilitate long-distance vesicle transport to the growing apex that constitutes one of the main driving forces of polarized growth. This study observed the spatial and temporal distribution of microtubules in growing Neurospora crassa hyphae in two and three dimensions. The fungal strain used expressed a normal growth pattern combined with the expression of green fluorescent protein (GFP) along the microtubules enabling their direct study with fluorescence and confocal microscopy. Time-lapse imaging revealed that the microtubules were dynamic, being assembled and disassembled at high rates as well as moving with the cytoplasmic flow. In the apical compartment, the filaments were arranged mostly parallel but not helical to the growth axis and appeared to determine the growth direction in close interaction with the Spitzenkörper. The microtubule distribution in subapical compartments was more random and their motility appeared to be driven by the cytoplasmic flow. However, this flow is affected by hyphal septa that act as partition walls with small connecting pores. Three-dimensional imaging showed that in order to pass through a septum, the filaments had to align parallel to the growth axis. The aim of this study was to attempt to reveal deeper insight into the role of microtubules in fungal growth thus confirming and challenging some suggestions proposed in previous literature.

We have investigated a confocal fluorescence detection system for 3D cultured mammalian cells in a microfluidic cell culture analog system. For direct measurement of metabolic changes in the microfluidic cell culture systems using 3D cell cultures, we constructed a compact and portable confocal fluorescence detection system based on discrete optical components. The confocal detection system was designed to provide a depth scan in situ without lateral imaging for fast scanning and acquisition of average fluorescence variation associated with a large number of cells in 3D cultures of microfluidic devices. The results were consistent with the data obtained by a standard confocal microscope and suggest the potential for monitoring cell dynamics in 3D cell-based assays.

Flow cytometry is a technology that allows a single cell or particle to be measured for a variety of characteristics, determined by looking at their properties while they flow in a liquid stream. High speed of flow and huge number of objects to be analyzed imposed some strict criteria on which methods can be used for analysis. All the known commercial instruments are currently using light scattering for particle sizing and fluorescence detection for chemical recognition. However, vibrational spectroscopy is the only non-invasive optical spectroscopy tool, which has proven to provide chemically-specific information about the interrogated sample. It is proposed that vibrational spectroscopy, based on nonlinear Raman scattering can be used to serve as an analytical tool for cytometry by providing rapid and accurate chemical recognition of flowing materials. To achieve a desired speed (>10,000 cell/particles per second), we have substantially upgraded our previous system for nonlinear Raman microspectroscopy. By increasing the size of the excitation volume to the size of a cell and by keeping the incident intensity at the same level, a dramatic increase of the nonlinear Raman signal is achieved. This allows high-quality vibrational spectra to be acquired within 10-100 microsecond from a single yeast cell without any observable damage to the irradiated cell. This is four orders of magnitude better than any previous attempts involving Raman microspectroscopy.

A highly sensitive technique for detection and quantification of protein-analyte binding interaction using spectral domain low coherence interferometric technique is implemented. Using this technique, it is possible to study real time interactions by quantifying the changes in optical path length caused by protein-analyte binding. The technique does not require elaborate sensor preparation and calibration. Any coverslip with certain thickness and capability to bind protein can work using this technique. For proof of principle, the interaction between IgG antigen and anti-IgG was studied and quantification of the accumulation of anti-IgG on sensor surface was demonstrated. Additionally, for biological relevance interactions between cancer biomarker EGFr and EGF was also studied and the preliminary results are presented. The potential applications of the label free technique include point-of-care screening of cancer biomarkers and tumor cell adhesion analysis.

Time-domain terahertz (THz) spectrometry has been used to analyze DNA hybridization state and its quantitation in a label-free manner. Time-resolved THz signal (or temporal signal) converted to frequency domain constitutes a signature of a given molecular "event" (e.g., a vibrational or a conformational state). The temporal signal provides a means of probing a molecular event in an appropriate time window. This is a unique ability of this technique because different molecular events exhibit different time response based on their physical and chemical nature. Conformational difference of a given molecule results in different signature with an appropriate time response that can be accurately probed by a terahertz temporal signal. In this work we discriminate between single stranded and double stranded 25-mer oligonucleotides via spectral signature. For each species, different peaks were identified; however, the peaks are distinctly different allowing an easy comparison. Additionally, temporal transmission spectra of the DNA specimens were collected at normal temperature and atmosphere. The peak value extracted from the temporal spectra exhibit a power law behavior over a region spanning from 13.6 femto-molar to 0.136 nano-molar. The results clearly demonstrate the ability of the spectrometer to detect a minute amount of biomolecules in a labelfree fashion. This capability can be used as a diagnostic tool, as well as for studying molecular reactions such as mutation.

This paper describes a new platform for quantitative intact proteomics, entitled Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis (CuTEDGE). The CuTEDGE technology utilizes differences in fluorescent lifetimes to subtract the confounding background fluorescence during in-gel detection and quantification of proteins, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily designed for image acquisition in 2-dimensional gel electrophoresis (2-DE), but is also applicable to 1-dimensional gel electrophoresis (1-DE), and proteins electroblotted to membranes. In a set of proof-of-principle measurements, we have evaluated the performance of the novel technology using the MicroTime 100 instrument (PicoQuant GmbH) in conjunction with the CyDye minimal labeling fluorochromes (GE Healthcare, Uppsala, Sweden) to perform differential gel electrophoresis (DIGE) analyses. The results indicate that the CuTEDGE technology provides an improvement in the dynamic range and sensitivity of detection of 3 orders of magnitude as compared to current state-of-the-art image acquisition instrumentation available for 2-DE (Typhoon 9410, GE Healthcare). Given the potential dynamic range of 7-8 orders of magnitude and sensitivities in the attomol range, the described invention represents a technological leap in detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.

We report a practical method for label-free quantification of specific molecules using spectroscopic imaging of sampleinduced phase shifts (for the detail, please see the Ref. [1]). Diffraction phase microscopy equipped with various wavelengths of light source is used to record wavelength-dependent phase images. We first perform dispersion measurements on pure solutions of single molecular species present in the cells, such as albumin and hemoglobin (Hb). With this prior calibration of molecular specific dispersion, we demonstrate the extraction of Hb concentration from individual red blood cells (RBCs). The end point of this study is non-invasive monitoring of physiological states of intact living cells.

We present a novel experimental setup to intravitally induce and monitor tissue lesions intravitally at a subcellular level in murine small intestinal mucosa. Using single 355-nm, 500-ps laser pulses coupled to a two-photon microscope, we induced optical breakdown with subsequent cavitation bubble formation in the tissue. Imaging was based on spectrally resolved two-photon excited tissue autofluorescence, while online-dosimetry of the induced microbubbles was done by a cw probe-beam scattering technique. From the scattering signal, the bubble size and dynamics could be deduced on a ns time scale. In turn, this signal could be used to control the damage size. This was important for reproducible production of minute effects in the tissue, despite strong biological variations in tissue response to pulsed laser irradiation. After producing local UV damage, cells appeared dark, probably due to destruction of mitochondria and loss of NAD(P)H fluorescence. Within 10 min after cell damage, epithelial cells adjacent to the injured area migrated into the wound to cover the denuded area, resulting in extrusion of the damaged cells from the epithelial layer. Using the nuclear acid stain propidium iodide, we could show that UV pulses induced cell membrane damage with subsequent necrosis, rather than apoptosis. For lesions without disruption of the basement membrane, we did not detect migration of immune cells toward the injured area within observation periods of up to 5 hours. This model will be used in further studies to investigate the intrinsic repair system and immune response to laserinduced lesions of intestinal epithelium in vivo.

We have demonstrated the high-speed confocal fluorescence lifetime imaging microscopy (FLIM) by analog mean-delay (AMD) method. The AMD method is a new signal processing technique for calculation of fluorescence lifetime and it is very suitable for the high-speed confocal FLIM with good accuracy and photon economy. We achieved the acquisition speed of 7.7 frames per second for confocal FLIM imaging. Here, the highest photon detection rate for one pixel was larger than 125 MHz and averaged photon detection rate was more than 62.5 MHz. Based on our system, we successfully obtained a sequence of confocal fluorescence lifetime images of RBL-2H3 cell labeled with Fluo-3/AM and excited by 4αPDD (TRPV channel agonist) within one second.

Noise reduction algorithms for improving Raman spectroscopy signals while preserving signal information were implemented. Algorithms based on Wavelet denoising and Kalman filtering are presented in this work as alternatives to the well-known Savitky-Golay algorithm. The Wavelet and Kalman algorithms were designed based on the noise statistics of real signals acquired using CCD detectors in dispersive spectrometers. Experimental results show that the random noise generated in the data acquisition is governed by sub-Poisson statistics. The proposed algorithms have been tested using both real and synthetic data, and were compared using Mean Squared Error (MSE) and Infinity Norm (L_∞) to each other and to the standard Savitky-Golay algorithm. Results show that denoising based on Wavelets performs better in both the MSE and (L_∞) the sense.

The latest development of commercial routine flow cytometers (FCM) is that they are equipped with three (blue, red, violet) or more lasers and many PMT detectors. Nowadays routine clinical instruments are capable of detecting 10 or more fluorescence colors simultaneously. Thereby, presenting opportunities for getting detailed information on the single cell level for cytomics and systems biology for improve diagnostics and monitoring of patients. The University Leipzig (Germany) recently started a cluster of excellence to study the molecular background of life style and environment associated diseases, enrolling 25000 individuals (LIFE). To this end the most comprehensive FCM protocol has to be developed for this study. We aimed to optimize fluorochrome and antibody combinations to the characteristics of the instrument for successful 10-color FCM. Systematic review of issues related to sampling, preparation, instrument settings, spillover and compensation matrix, reagent performance, and general principles of panel construction was performed. 10-color FCM enables for increased accuracy in cell subpopulation identification, the ability to obtain detailed information from blood specimens, improved laboratory efficiency, and the means to consistently detect major and rare cell populations. Careful attention to details of instrument and reagent performance allows for the development of panels suitable for screening of samples from healthy and diseased donors. The characteristics of this technique are particularly well suited for the analysis of broad human population cohorts and have the potential to reach the everyday practice in a standardized way for the clinical laboratory.

In advanced cytometry, a fundamental challenge for rapid specific detection of rare-event micro-organisms is the autofluorescence noise from the complex biological samples. Time-gated luminescence can effectively discriminate labeled cells from autofluorescence background. Recently, a real-time true-colour time-gated luminescence microscopy system has been developed based on the synchronization of a solid-state excitation source and a super-fast optical shutter. We also developed a variety of ultra-bright silica nano-biolabels with multiple luminescence colours and controllable lifetimes in microsecond range. These developments allowed the development of an advanced cell analysis system for real-time background-free imaging and rare-event counting of microsecond-lifetime multi-colour labelled water-borne pathogens.

Problem: Previous images of time-gated luminescence have been obtained with a cooled CCD camera by digitally summing a series of sequential images. The data acquisition rate of approximately 10 one millisecond exposure images per second was rate limiting and too slow for standard research and clinical use. An annoying undulating background was present, which could not be totally removed by subtraction of an unexposed, control image. Solution: An analog approach to this problem is to use an interline transfer, electronically shuttered camera. After each exposure, the storage line is not readout; instead, the electrons from the acquisition pixels are transferred to the storage pixels and thus are added to those previously stored. The length of the exposure is limited by the capacity of the storage pixels and the rate of generation of background (noise) electrons. This electronic concept was tested with a Point Grey Dragonfly2 640 by 480 pixel monochrome camera equipped with a Sony 1/3" progressive interline scan, electronically shuttered CCD, which since it did not have any cooling, was operated at room temperature. Pulsed excitation was from a Nichia UV LED. Results: Five and 0.5 micron uniform europium complex stained microspheres could at room temperature be imaged with time-gated excitation and acquisition times of 1 millisecond each and analog summation of 50 images. Conclusion: The analog integration solution apparently works; however, a cooled scientific grade camera with the same capacity for multiple transfers into storage pixels would be better suited for use with dimmer luminescent objects.

For description of cellular phenotypes and physiological states new developments are needed. Axetris' impedance flow cytometer (IFC) (Leister) is a new promising label-free alternative to fluorescence-based flow cytometry (FCM). IFC measures single cells at various frequencies simultaneously. The frequencies used for signal acquisition range from 0.1 to 20 MHz. The impedance signal provides information about cell volume (< 1 MHz), membrane capacitance (~1-4 MHz) and cytoplasmic conductivity (4-10 MHz), parameters directly related to the physiological conditions of single cells. In MCF-7 cell viability experiments, cells were treated with cytotoxic agents to induce cell death. Impedance analysis showed discrimination between viable and dead cells. This was clearly visible at 4 MHz suggesting that differentiation was possible based on cell membrane capacitance. Changes in cell membrane potential were also analysed by IFC. RN22 cells were loaded with membrane potential sensitive dye (DiBAC4). The cells were then treated with the ionophore valinomycin. Changes in membrane potential were detectable at the level of cytoplasm conductivity (>4 MHz) and membrane capacitance (1-4 MHz). Our data indicate that IFC can be a valuable alternative to conventional FCM for various applications in the field of cell death and physiology. The work will be extended to address further potential applications of IFC in biotechnology and biomedical cell analysis, as well as in cell sorting.

We have developed a fundamentally new type of cytometer to track the statistics of dynamic molecular interactions in hundreds of individual live cells within a single experiment. This entirely new high-throughput experimental system, which we have named Cyto•IQ, reports statistical, rather than image-based data for a large cellular population. Like a flow cytometer, Cyto•IQ rapidly measures several fluorescent probes in a large population of cells to yield a reduced statistical model that is matched to the experimental goals set by the user. However, Cyto•IQ moves beyond flow cytometry by tracking multiple probes in individual cells over time. Using adaptive learning algorithms, we process data in real time to maximize the convergence of the statistical model parameter estimators. Software controlling Cyto•IQ integrates existing open source applications to interface hardware components, process images, and adapt the data acquisition strategy based on previously acquired data. These innovations allow the study of larger populations of cells, and molecular interactions with more complex dynamics, than is possible with traditional microscope-based approaches. Cyto•IQ supports research to characterize the noisy dynamics of molecular interactions controlling biological processes.

Patch clamping is used in electrophysiology to study single or multiple ion channels in cells. Multiple micropipettes are used as electrodes to collect data from several cells. Placement of these electrodes is a time consuming and complicated task due to the lack of depth perception, limited view through the microscope lens and the possibility of collisions between micro-pipettes. To aid in this process, a computer-assisted approach is developed using image processing techniques applied to images obtained through the microscope. Image processing algorithms are applied to perform autofocusing, relative depth estimation, distance estimation and tracking of the micro-pipettes in the images without making any major changes in the existing patch clamp equipment. An autofocusing algorithm with a micrometer precision is developed and the relative depth estimation is performed based on autofocusing. A micro-pipette tip detection algorithm is developed which can be used to initialize or reset the tracking algorithm and to calibrate the system by registering the relative image and micro-manipulator coordinates. An image-based tracking algorithm is also developed to track a micro-pipette tip in real time. The real-time tracking data is then used for visual servoing the micro-pipette tips and updating the calibration information.

The Cell Transformation Assay (CTA) is one of the promising in vitro methods used to predict human carcinogenicity. The neoplastic phenotype is monitored in suitable cells by the formation of foci and observed by light microscopy after staining. Foci exhibit three types of morphological alterations: Type I, characterized by partially transformed cells, and Types II and III considered to have undergone neoplastic transformation. Foci recognition and scoring have always been carried visually by a trained human expert. In order to automatically classify foci images one needs to implement some image understanding algorithm. Herewith, two such algorithms are described and compared by performance. The supervised classifier (as described in previous articles) relies on principal components analysis embedded in a training feedback loop to process the morphological descriptors extracted by "spectrum enhancement" (SE). The unsupervised classifier architecture is based on the "partitioning around medoids" and is applied to image descriptors taken from histogram moments (HM). Preliminary results suggest the inadequacy of the HMs as image descriptors as compared to those from SE. A justification derived from elementary arguments of real analysis is provided in the Appendix.

Early stages of tumor angiogenesis can be modeled by various in vitro cultures in which endothelial cells (ECs) form networks that are considered to mimic the vascularization of tumors in vivo. Image based quantification of EC culture model is a useful method for effective characterization of early stage in vitro vasculogenesis and the effects of pro and anti-angiogenesis reagents. We propose an image analysis method to quantify the EC tube formation in 2D cultures. The method segments images by high pass filtering in Fourier space, followed by thresholding and a skeletonization and pruning process to generate the binary skeleton image of the cell patterns in culture. Several quantities such as the network entropy (NE), the node number, total number of chords, total and average chord length were used to quantify the evolution of EC tubes. The automatic measurement of chord length was validated against manual measurement, achieving an R² value of 0.953, and was used to assay for tubal extension as a function of increasing VEGF concentration. Measurements of NE, node number, chord lengths were demonstrated on ECs network-like patterns in culture.

Charge-Coupled Device (CCD) detectors are becoming more popular in spectroscopy instrumentation. In spite of technological advances, spurious signals and noise are unavoidable in Raman spectroscopes. In general, the noise comes from two major sources, impulsive noise caused by high energy radiation from local or extraterrestrial sources (cosmic rays), and noise produced in Raman backscattering estimation. In this work, two algorithms for impulsive noise removal are presented, based in spectral and spatial features of the noise. The algorithms combine pattern recognition and classical filtering techniques to identify the impulses. Once an impulse has been identified, it is removed and substituted with data points having similar statistical properties as the surrounding data.

We designed and fabricated a flow cytometry work bench for the purpose of testing optical schemes utilized in two specialized flow cytometry techniques. Fluorescent spectroscopy and multi-angle scattering of laser light has potential for generating novel classes of cell population information. The acquisition of full fluorescence spectrum of dye bound to living cells may associate specific function to spectral shift. Similarly, detection of 2 discrete angles of light scatter was shown to describe internal structures of cells based on refractive and diffractive properties. We utilized a flow cytometry electro- cells based on refractive and diffractive properties. We utilized a flow cytometry electro- optical test bench to develop and describe alternate light collection schemes for these two techniques. Mouse bone marrow was chosen as the subject of analysis because it contains a consistent mix of cells that possess distinct biophysical properties. More importantly, these cells are the source of various biological systems that are targeted by environmental stress and clinical disease.

We experimentally characterized a novel Angular Domain Spectroscopic Imaging (ADSI) technique for the detection and characterization of optical contrast abnormalities in turbid media. The new imaging system employs silicon micromachined angular filtering methodology, which has high angular selectivity for photons exiting the turbid medium. The angular filter method offers efficient scattered light suppression at moderate levels of scattering (i.e. up to 6 reduced mean free paths). An ADSI system was constructed from a broadband light source, an Angular Filter Array (AFA), and an imaging spectrometer. The free-space collimated broadband light source was used to trans-illuminate a turbid sample over a wide range of wavelengths in the near infrared region of the spectrum. The imaging spectrometer decomposed the output of the AFA into hyperspectral images representative of spatial location and wavelength. It collected and angularly filtered a line image from the object onto the CCD camera with the spatial information displayed along one axis and wavelength information along the other. The ADSI system performance was evaluated on tissue-mimicking phantoms as well as fresh chicken breast tissue. Collected images with the ADSI displayed differences in image contrast between different tissue types.

Spectral imaging involves capturing images at multiple wavelengths resulting in a data cube (x, y, λ) that allows materials to be identified by its spectral signature. While hyperspectral imagers can provide high spectral resolution, they also have major drawbacks such as cost, size, and the copious amounts of data in the image cube. Typically, the complete hyperspectral data cube provides little additional information compared to only 3-8 discrete (multiwavelength) imaging bands. We present two new approaches and related technologies where we are able to acquire spectral imaging data stacks quickly and cost-effectively. Our two spectral imaging systems represent different approaches integrated with standard CCD and CMOS imagers: sequential rotating filter wheels (RFWs) and lithographically patterned dichroic filter arrays (DFAs). The RFW approach offers the ability for rapid configuration of a spectral system, and a whole new level of self-contained image acquisition, processing and on-board display. The DFA approach offers the potential for ultra compact imagers with acquisition of images of multiple wavelengths simultaneously, while still allowing for processing and display steps to be built into the camera. Both approaches lend themselves production of multi-wavelength/spectral imaging systems with differing features and advantages.

Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.

We report a technique which can overcome these drawbacks, but maintains the advantage of phase microscopy - high contrast live cell imaging and 3D imaging (For the detail, please see the Ref. [1]). A speckle beam of a complex spatial pattern is used for illumination to reduce fixed pattern noise and to improve optical sectioning capability. By recording of the electric field of speckle, we demonstrate high contrast 3D live cell imaging without the need for axial scanning - neither objective lens nor sample stage. This technique has great potential in studying biological samples with improved sensitivity, resolution and optical sectioning capability.

Our digital holographic (DH) approach can be used to study tissue structures both in vitro and in vivo. This DHM architecture can produce three color microscopic 3D and 4D (video) images. We record 3 color (RGB) holograms with single exposures, and the perfect compensation of color crosstalk is solved. An in-line holographic setup and reconstruction algorithms are presented with demonstrative simulations and experimentally captured and numerically reconstructed images. Comparing the individually reconstructed color images with each other can provide information both for recognition of different types of cells or microorganisms, and for diagnostic purposes as well. Experimental example is given observing microscopic hydro-biological organisms using a color digital holographic microscope.

In optical microscopy, the polarization state of the focal field strongly influences formed images due to its interactions with the sample and the effective focal spot size. We demonstrate experimentally that control over the spatial profile of the focal field polarization state improves spatial resolution in laser-scanning third harmonic generation (THG) microscopy. The focal field is manipulated by imaging a spatial light modulator to the focal plane of a moderate-numerical aperture microscope. The resolution enhancement arises from exploiting the suppression, in isotropic media, of THG for circularlypolarized field polarization. By synthesizing a focal field whose polarization state changes from linear at the beam center to circular beyond radius rs, we quench THG beyond rs. A transverse spatial resolution of up to 2 times is demonstrated. Targeted manipulation necessitates measurement techniques that allow us to determine of the focal field polarization state. We develop two such techniques to characterize the field. We use a nano-particle with known third-order susceptibility to localize THG scattering to a small focal volume. Scanning this nano-probe through the focal volume of the microscope allows for complete reconstruction of the vector point spread function. Under moderate focusing conditions, where the recorded THG signal is dominated by the incident paraxial polarization component, the spatial polarization state is determined non-iteratively via three linear-polarization projection THG measurements. Under tight focusing conditions, polarization scrambling occurs such that the input and focal fields are dissimilar, and we introduce an algorithm for focal field retrieval through the collection of far-field THG images.

The investigation of the nanostructures and hydrophobic properties of cancer cell membranes is of importance for elucidating the plasma membrane roles in protein folding, membrane fusion, and cell adhesion that are directly related to cancer cell biophysical properties, such as aggressive growth and migration. On the other hand, the chemical component analysis of the cancer cell membrane could be potentially applied in the clinical diagnosis of cancer by the identification of specific biomarker receptors expressed on cancer cell surfaces. In the present work, a combined atomic force microscopy (AFM) and Raman microspectroscopy technique was applied to detect the difference in nanomechanics and membrane chemical components between two cancer cell lines, human lung adenocarcinoma epithelial cells (A549) and human breast cancer cells (MDA-MB-435 with and without expression of BRMS1 metastasis suppressor). The membrane surface adhesion forces for these cancer cells acquired in culture medium were measured using AFM at 0.478±0.091 nN for A549 cells, 0.253±0.070 nN for 435 cells, and 1.114±0.281 nN for 435/BRMS1 cells, and the cell spring constant was measured at 2.62±0.682 mN/m for A549 cells, 2.105±0.691 mN/m for 435 cells, and 5.448±1.081 mN/m for 435/BRMS1 cells. Raman spectral analysis indicated similar peaks between the A549 cells and the breast cancer cell lines 435 and 435/BRMS1 including ~720 cm^-1 (guanine band of DNA), 940 cm^-1 (skeletal mode polysaccharide), 1006 cm^-1 (symmetric ring breathing phenylalanine), and 1451 cm-1 (CH deformation). Slight variations were observed between ~780 - 985 cm^-1 (DNA/RNA and proteins) and 1035 - 1210 cm^-1 (lipid and proteins).

Measurement of cerebral perfusion is important for study of various brain disorders such as stroke, epilepsy, and vascular dementia; however, efficient and convenient methods which can provide quantitative information about cerebral blood flow are not developed. Here we propose an optical imaging method using time-series analysis of dynamics of indocyanine green (ICG) fluorescence to generate cerebral blood flow maps. In scalp-removed mice, ICG was injected intravenously, and 740nm LED light was illuminated for fluorescence emission signals around 820nm acquired by cooled-CCD. Time-lapse 2-dimensional images were analyzed by custom-built software, and the maximal time point of fluorescent influx in each pixel was processed as a blood flow-related parameter. The generated map exactly reflected the shape of the brain without any interference of the skull, the dura mater, and other soft tissues. This method may be further applicable for study of other disease models in which the cerebral hemodynamics is changed either acutely or chronically.

Even though electrical stimulation is generally used for induction of smooth muscle cell contraction, it is very hard to obtain fine control and also very invasive for inserting electrodes. Herein, we developed a new optical technology to control smooth muscle cell contraction. This optical method using femtosecond pulsed laser (FSPL) has advantage of focused stimulation and fine control of stimulation intensity. Upon brief exposure to FSPL, smooth muscle cells showed a rapid increase of intracellular calcium levels followed by cell contraction. Collectively, we suggest that FSPL can be a useful tool for control of smooth muscle cell contraction.

Autophagy is a protein degradation process in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon the induction of autophagy, a double membrane autophagosome forms around cytoplasmic components and delivers them to the vacuole for degradation. In plants, autophagy has been shown previously to be induced during abiotic stresses including oxidative stress. Cd, as a toxicity heavy metal, resulted in the production of reactive oxygen species (ROS). In this paper, we demonstrated that ROS contributed to the induction of autophagy in Cd-stressed Arabidopsis thaliana. However, pre-incubation with ascorbic acid (AsA, antioxidant molecule) and catalase (CAT, a H₂O₂-specific scavenger) decreased the ROS production and the number of autolysosomal-like structures. Together our results indicated that the oxidative condition was essential for autophagy, as treatment with AsA and CAT abolished the formation of autophagosomes, and ROS may function as signal molecules to induce autophagy in abiotic stress.

Autophagy appears to be a highly conserved process from unicellular to multicellular eukaryotes which contributes to the equilibrium of intracelluar environment. While it would be harmful to the cells when it is excessive by inducing programmed cell death (PCD). It is a protein degradation process in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Previous studies have demonstrated autophagy can be induced during abiotic or biotic stresses. salicylic acid (SA) and methyl salicytic (MeSA) are endogenous signal molecules. We found SA and MeSA can induce autophagy in Arabidopsis thaliana respectively. While autophagy was not induced by SA or MeSA in tobacco suspension cells under the same concentration and period. The differences in stuctures or physiological states may contribute to the results.

Noninvasive measurement technique to obtain tissue optical properties such as the scattering coefficient μs and the anisotropy factor g using optical coherence tomography (OCT) scattering model which based on the Extended Huygens-Fresnel principle is developed in our paper. Older and younger mouse-skin are as animal model to compare its scattering coefficient μs and the anisotropy factor g, the outcome shows that scattering coefficient μs is increased with the age of mouse-skin. Furthermore, we have made age's mouse-skin into H.E stain slices; the result of its morphology is consistent with the OCT imaging and OCT-EHF principle. All of that have provided the theoretical basis which to the research on photo-aging skin and photo-rejuvenation.

In-vivo confocal microscope technology can be applied to the medical imaging diagnosis and new drug development. We present an in-vivo confocal microscope that can acquire a reflection image and a fluorescence image simultaneously and independently. To obtain reflection confocal images, we used a linearly polarized diode laser with the wavelength of 830 nm. To acquire fluorescence confocal images, we used two diode lasers with the wavelength of 488 nm and 660 nm, respectively. Because of a broad wavelength bandwidth from visible (488 nm) to near-IR (830 nm), we designed and optimized the optical system to reduce various optical aberrations. With the developed in-vivo confocal microscope, we performed ex-vivo cell imaging and in-vivo imaging of the human skin.

Wild type Arabidopsis thaliana (ecotype Columbia) and Thellungiella were used as experimental material in this artile. The leaves of the Arabidopsis and the Thellungiella were treated with different concentrations of NaCl. The fluorescence emission spectra, the chlorophyll fluorescence and the delayed fluorescence were detected respectively. We found that there was an obvious change in the photosynthetic efficiency of PS II and the DF intensity of the Arabidopsis leaves with different concentrations of NaCl treatment. However, there wasnot an obvious change in the photosynthetic efficiency of PS II and the DF intensity of the Thellungiella leaves with 100mmol/L and 200mmol/L NaCl treatment. While it also showed that there came to be an obvious stress of the Thellungiella leaves with 300mmol/L NaCl treatment. The fast chlorophyll fluorescence induction dynamics analysis revealed that the photosynthetic efficiency of PS II of the Arabidopsis leaves had a sharp decline along with the 100-200 mmol/L NaCl treatment, While the Thellungiella leaves showed a strong tolerances to salt stress.

Rheumatoid Arthritis is a systemic chronic inflammatory disease, recurrent and systemic, initiated by autoantibodies and maintained by inflammatory mechanisms cellular applicants. The evaluation of this disease to promote early diagnosis, need an associations of many tools, such as clinical, physical examination and thorough medical history. However, there is no satisfactory consensus due to its complexity. In the present work, confocal Raman spectroscopy was used to evaluate the biochemical composition of human serum of 40 volunteers, 24 patients with rheumatoid arthritis presenting clinical signs and symptoms, and 16 healthy donors. The technique of latex agglutination for the polystyrene covered with human immunoglobulin G and PCR (protein c-reactive) was performed for confirmation of possible false-negative results within the groups, facilitating the statistical interpretation and validation of the technique. This study aimed to verify the changes for the characteristics Raman peaks of biomolecules such as immunoglobulins amides and protein. The results were highly significant with a good separation between groups mentioned. The discriminant analysis was performed through the principal components and correctly identified 92% of the donors. Based on these results, we observed the behavior of arthritis autoimmune, evident in certain spectral regions that characterize the serological differences between the groups.

We present a tomographic imaging system that is applied a continuous phase shifting interferometry scheme to digital holographic microscopy (DHM). The proposed scheme achieves en-face tomographic image from digitally recorded original hologram by 2-D sensor array. Although images obtained at out-of-focus position, the application of integrating four bucket technique to digital hologram produces refocused en-face image with showing clear field of view. The proposed technique has advantages such as reduced phase errors and faster acquisition speed when it compared with conventional discrete phase stepping method. The performance of the system is demonstrated with presenting of the images on a scratched mirror surface and of an USAF resolution target. The reconstructed images are compared with conventional microscopic images, which reveal good aggrements. We believe that the proposed method enables tomographic imaging of biological samples with providing reduced noise level and improved imaging speed.

In cell biology studies it is often important to avoid the damaging effects caused by fluorescent stains or UV-light. Immersion Mirau Interferometry (IMI) is an epi-illumination label-free imaging technique developed at the Columbia University Radiological Research Accelerator Facility. It is based on the principles of phase-shifting interferometry (PSI) and represents a novel approach for interferometric imaging of living cells in medium. To accommodate the use of medium, a custom immersion Mirau interferometric attachment was designed and built in-house. The space between the reference mirror and the beam splitter is filled with liquid to ensure identical optical paths in the test and reference arms. The interferometer is mountable onto a microscope objective. The greatest limitation of standard PSI is the sensitivity to environmental vibrations, because it requires consecutive acquisition of several interferograms. We are developing Simultaneous Immersion Mirau Interferometry (SIMI), which facilitates simultaneous acquisition of all interferograms and eliminates the effects of vibration. Polarization optics, incorporated into the design, introduces a phase delay to one of the components of the test beam. This enables simultaneous creation and spatial separation of two interferograms, which are combined with the background image to reconstruct the intensity map of the specimen. Our results of imaging live and fixed cells with IMI and SIMI show that this system produces images of a quality that is sufficient to perform targeted cellular irradiation experiments.