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Sample records for multiphoton spectral imaging

  1. High speed multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Li, Yongxiao; Brustle, Anne; Gautam, Vini; Cockburn, Ian; Gillespie, Cathy; Gaus, Katharina; Lee, Woei Ming

    2016-12-01

    Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 × 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice.

  2. Quantitative multiphoton imaging

    NASA Astrophysics Data System (ADS)

    König, Karsten; Weinigel, Martin; Breunig, Hans Georg; Uchugonova, Aisada

    2014-02-01

    Certified clinical multiphoton tomographs for label-free multidimensional high-resolution in vivo imaging have been introduced to the market several years ago. Novel tomographs include a flexible 360° scan head attached to a mechanooptical arm for autofluorescence and SHG imaging as well as a CARS module. Non-fluorescent lipids and water, mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen can be imaged in vivo with submicron resolution in human skin. Sensitive and rapid detectors allow single photon counting and the construction of 3D maps where the number of detected photons per voxel is depicted. Intratissue concentration profiles from endogenous as well exogenous substances can be generated when the number of detected photons can be correlated with the number of molecules with respect to binding and scattering behavior. Furthermore, the skin ageing index SAAID based on the ratio elastin/collagen as well as the epidermis depth based on the onset of SHG generation can be determined.

  3. Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

    DTIC Science & Technology

    2007-03-01

    non-linear imaging modality( 2 ), which combines current state of the art imaging techniques (fluorescence spectroscopy and fluorescence lifetime...calibrated and helped us to confirm the fluorescence source from living cells under our imaging conditions (789nm 2 -photon excitation) is indeed NADH... Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.” Exp Cell Res 309( 2 ): 390-6. DeMali, K. A. and Burridge, K. (2003

  4. Multiphoton imaging of freezing and heating effects in plant leaves.

    PubMed

    Breunig, Hans Georg; Tümer, Fatma; König, Karsten

    2013-08-01

    Thermally-induced changes in Arabidopsis thaliana leaves were investigated with a novel cryo microscope by multiphoton, fluorescence lifetime and spectral imaging as well as micro spectroscopy. Samples were excited with fs pulses in the near-infrared range and cooled/heated in a cryogenic chamber. The results show morphological changes in the chloroplast distribution as well as a shift from chlorophyll to cell-wall fluorescence with decreasing temperature. At temperatures below -40 °C, also second harmonic generation was observed. The measurements illustrate the suitability of multiphoton imaging to investigate thermally-induced changes at temperatures used for cryopreservation as well as for basic investigations of thermal effects on plant tissue in general.

  5. Multiphoton imaging of renal regulatory mechanisms.

    PubMed

    Peti-Peterdi, János; Toma, Ildikó; Sipos, Arnold; Vargas, Sarah L

    2009-04-01

    Most physiological functions of the kidneys, including the clearance of metabolic waste products, maintenance of body fluid, electrolyte homeostasis, and blood pressure, are achieved by complex interactions between multiple renal cell types and previously inaccessible structures in many organ parts that have been difficult to study. Multiphoton fluorescence microscopy offers a state-of-the-art imaging technique for deep optical sectioning of living tissues and organs with minimal deleterious effects. Dynamic regulatory processes and multiple functions in the intact kidney can be quantitatively visualized in real time, noninvasively, and with submicron resolution. This article reviews innovative multiphoton imaging technologies and their applications that provided the most complex, immediate, and dynamic portrayal of renal function-clearly depicting as well as analyzing the components and mechanisms involved in renal (patho)physiology.

  6. Multiphoton imaging of renal tissues in vitro.

    PubMed

    Peti-Peterdi, János

    2005-06-01

    The highly inhomogeneous and light-scattering structure of living renal tissue makes the application of conventional imaging techniques more difficult compared with other parenchymal organs. On the other hand, key physiological processes of the kidney, such as regulation of glomerular filtration, hemodynamics, concentration, and dilution, involve complex interactions between multiple cell types and otherwise inaccessible structures that necessitate visual approaches. An ideal solution is multiphoton excitation fluorescence microscopy, a state-of-the-art imaging technique superior for deep optical sectioning of living tissue samples. Here, we review the basics and advantages of multiphoton microscopy and provide examples for its application in renal physiology using dissected cortical and medullary tissues in vitro. In combination with microperfusion techniques, the major functions of the juxtaglomerular apparatus, tubuloglomerular feedback and renin release, can be studied with high spatial and temporal resolution. Salt-dependent changes in macula densa cell volume, vasoconstriction of the afferent arteriole, and activity of an intraglomerular precapillary sphincter composed of renin granular cells are visualized in real time. Release and tissue activity of renin can be studied on the individual granule level. Imaging of the living inner medulla shows how interstitial cells interconnect cells of the vasa recta, loop of Henle, and collecting duct. In summary, multiphoton microscopy is an exciting new optical sectioning technique that has great potential for numerous future developments and is ideal for applications that require deep optical sectioning of living tissue samples.

  7. Live cell imaging by multifocal multiphoton microscopy.

    PubMed

    Straub, M; Lodemann, P; Holroyd, P; Jahn, R; Hell, S W

    2000-10-01

    Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.

  8. Multiphoton imaging with a nanosecond supercontinuum source

    NASA Astrophysics Data System (ADS)

    Lefort, Claire; O'Connor, Rodney P.; Blanquet, Véronique; Baraige, Fabienne; Tombelaine, Vincent; Lévêque, Philippe; Couderc, Vincent; Leproux, Philippe

    2016-03-01

    Multiphoton microscopy is a well-established technique for biological imaging of several kinds of targets. It is classically based on multiphoton processes allowing two means of contrast simultaneously: two-photon fluorescence (TPF) and second harmonic generation (SHG). Today, the quasi exclusive laser technology used in that aim is femtosecond titanium sapphire (Ti: Sa) laser. We experimentally demonstrate that a nanosecond supercontinuum laser source (STM-250-VIS-IR-custom, Leukos, France; 1 ns, 600-2400 nm, 250 kHz, 1 W) allows to obtain the same kind of image quality in the case of both TPF and SHG, since it is properly filtered. The first set of images concerns the muscle of a mouse. It highlights the simultaneous detection of TPF and SHG. TPF is obtained thanks to the labelling of alpha-actinin with Alexa Fluor® 546 by immunochemistry. SHG is created from the non-centrosymmetric organization of myosin. As expected, discs of actin and myosin are superimposed alternatively. The resulting images are compared with those obtained from a standard femtosecond Ti: Sa source. The physical parameters of the supercontinuum are discussed. Finally, all the interest of using an ultra-broadband source is presented with images obtained in vivo on the brain of a mouse where tumor cells labeled with eGFP are grafted. Texas Red® conjugating Dextran is injected into the blood vessels network. Thus, two fluorophores having absorption wavelengths separated by 80 nm are imaged simultaneously with a single laser source.

  9. Multiphoton microscopy as a diagnostic imaging modality for lung cancer

    NASA Astrophysics Data System (ADS)

    Pavlova, Ina; Hume, Kelly R.; Yazinski, Stephanie A.; Peters, Rachel M.; Weiss, Robert S.; Webb, Watt W.

    2010-02-01

    Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on twophoton excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.

  10. Studies on wide-field-of-view multiphoton imaging using the flexible clinical multiphoton tomograph MPTflex

    NASA Astrophysics Data System (ADS)

    Weinigel, Martin; Breunig, Hans Georg; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; König, Karsten

    2012-03-01

    Multiphoton imaging systems are capable of high-resolution 3-D image acquisition of deep tissue. A first commercially available CE-certified biomedical system for subcelluar resolution of human skin has been launched by JenLab company with the DermaInspectR in 2002. The demand for more flexibility caused the development of the MPTflexR, which provides an increased flexibility and accessibility especially for clinical and cosmetic examinations. However the high resolution of clinical multiphoton tomographs are adherent with a small field-of-view (FOV) of about 360×360μm2. Especially time-consuming is the relocation of areas of interest (AOI) like lesions, sweat glands or hair shafts during a multiphoton examination. This limitation can be be overcome by macroscopic large-area (wide-field-ofview) multiphoton tomography, which is tested first within this work.

  11. Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Salomonnson, Emma; Mihalko, Laura Anne; Verkhusha, Vladislav V.; Luker, Kathryn E.; Luker, Gary D.

    2012-09-01

    Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the state-of-the-art technology for longitudinal imaging studies in tissues and living animals. Successful analysis of separate cell populations or signaling events by intravital microscopy requires optimal pairing of multiphoton excitation wavelengths with spectrally distinct fluorescent proteins. While prior studies have analyzed two photon absorption properties of isolated fluorescent proteins, there is limited information about two photon excitation and fluorescence emission profiles of fluorescent proteins expressed in living cells and intact tissues. Multiphoton microscopy was used to analyze fluorescence outputs of multiple blue, green, and red fluorescent proteins in cultured cells and orthotopic tumor xenografts of human breast cancer cells. It is shown that commonly used orange and red fluorescent proteins are excited efficiently by 750 to 760 nm laser light in living cells, enabling dual color imaging studies with blue or cyan proteins without changing excitation wavelength. It is also shown that small incremental changes in excitation wavelength significantly affect emission intensities from fluorescent proteins, which can be used to optimize multi-color imaging using a single laser wavelength. These data will direct optimal selection of fluorescent proteins for multispectral two photon microscopy.

  12. Multiphoton imaging of biological samples during freezing and heating

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2014-02-01

    We applied multiphoton microscopic imaging to observe freezing and heating effects in plant- and animal cell samples. The experimental setups consisted of a multiphoton imaging system and a heating and cooling stage which allows for precise temperature control from liquid nitrogen temperature (-196°C 77 K) up to +600°C (873 K) with heating/freezing rates between 0.01 K/min and 150 K/min. Two multiphoton imaging systems were used: a system based on a modified optical microscope and a flexible mobile system. To illustrate the imaging capabilities, plant leafs as well as animal cells were microscopically imaged in vivo during freezing based on autofluorescence lifetime and intensity of intrinsic molecules. The measurements illustrate the usefulness of multiphoton imaging to investigate freezing effects on animal and plant cells.

  13. Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging.

    PubMed

    Cua, Michelle; Wahl, Daniel J; Zhao, Yuan; Lee, Sujin; Bonora, Stefano; Zawadzki, Robert J; Jian, Yifan; Sarunic, Marinko V

    2016-09-07

    Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems.

  14. Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging

    PubMed Central

    Cua, Michelle; Wahl, Daniel J.; Zhao, Yuan; Lee, Sujin; Bonora, Stefano; Zawadzki, Robert J.; Jian, Yifan; Sarunic, Marinko V.

    2016-01-01

    Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems. PMID:27599635

  15. Coherence-Gated Sensorless Adaptive Optics Multiphoton Retinal Imaging

    NASA Astrophysics Data System (ADS)

    Cua, Michelle; Wahl, Daniel J.; Zhao, Yuan; Lee, Sujin; Bonora, Stefano; Zawadzki, Robert J.; Jian, Yifan; Sarunic, Marinko V.

    2016-09-01

    Multiphoton microscopy enables imaging deep into scattering tissues. The efficient generation of non-linear optical effects is related to both the pulse duration (typically on the order of femtoseconds) and the size of the focused spot. Aberrations introduced by refractive index inhomogeneity in the sample distort the wavefront and enlarge the focal spot, which reduces the multiphoton signal. Traditional approaches to adaptive optics wavefront correction are not effective in thick or multi-layered scattering media. In this report, we present sensorless adaptive optics (SAO) using low-coherence interferometric detection of the excitation light for depth-resolved aberration correction of two-photon excited fluorescence (TPEF) in biological tissue. We demonstrate coherence-gated SAO TPEF using a transmissive multi-actuator adaptive lens for in vivo imaging in a mouse retina. This configuration has significant potential for reducing the laser power required for adaptive optics multiphoton imaging, and for facilitating integration with existing systems.

  16. Multiphoton, confocal, and lifetime microscopy for molecular imaging in cartilage

    NASA Astrophysics Data System (ADS)

    Wachsmann-Hogiu, Sebastian; Krakow, Deborah; Kirilova, Veneta T.; Cohn, Daniel H.; Bertolotto, Cristina; Acuna, Dora; Fang, Qiyin; Krivorov, Nikola; Farkas, Daniel L.

    2005-03-01

    It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.

  17. Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G.-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

    2016-06-01

    Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

  18. Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging

    PubMed Central

    Wang, Taejun; Jang, Won Hyuk; Lee, Seunghun; Yoon, Calvin J.; Lee, Jun Ho; Kim, Bumju; Hwang, Sekyu; Hong, Chun-Pyo; Yoon, Yeoreum; Lee, Gilgu; Le, Viet-Hoan; Bok, Seoyeon; Ahn, G-One; Lee, Jaewook; Gho, Yong Song; Chung, Euiheon; Kim, Sungjee; Jang, Myoung Ho; Myung, Seung-Jae; Kim, Myoung Joon; So, Peter T. C.; Kim, Ki Hean

    2016-01-01

    Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence. PMID:27283889

  19. MULTIPHOTON IMAGING CAN BE USED FOR MICROSCOPIC EXAMINATION OF INTACT HUMAN GASTROINTESTINAL MUCOSA EX VIVO

    PubMed Central

    Rogart, Jason N.; Nagata, Jun; Loeser, Caroline S.; Roorda, Robert D.; Aslanian, Harry; Robert, Marie E.; Zipfel, Warren R.; Nathanson, Michael H.

    2008-01-01

    Background & Aims The ability to observe cellular and subcellular detail during routine endoscopy is a major goal in the development of new endoscopic imaging techniques. Multiphoton microscopy, which relies on nonlinear infared optical processes, has the potential to identify cellular details by excitation of endogenous fluorescent molecules. We examined the feasibility of using multiphoton microscopy to characterize mucosal histology in the human gastrointestinal tract. Methods A multiphoton microscope was used to determine the optimal excitation wavelength for examination of gastrointestinal mucosa. Fresh, unfixed, and unstained biopsy specimens obtained during routine endoscopy in human subjects were then examined by confocal microscopy and multiphoton microscopy. Multiphoton images also were compared to standard H&E images obtained from paired biopsy specimens. A prototype miniaturized multiphoton probe was used to examine intact rat colon. Results Peak multiphoton autofluorescence intensity was detected in mucosa excited at 735 nm. Multiphoton microscopic examination of unstained biopsy specimens revealed improved cellular detail relative to either unstained or stained specimens examined by confocal imaging. Resolution of structures such as epithelial nuclei, goblet cells, and interstitial fibers and cells was comparable to what was obtained using standard H&E histology. Similar findings were observed when using a prototype miniaturized multiphoton probe. Conclusions Multiphoton microscopy can be used to examine gastrointestinal mucosa at the cellular level, without the need for fluorescent dyes. The construction of a multiphoton endomicroscope could therefore provide a practical means of performing “virtual biopsies” during the course of routine endoscopy, with advantages over currently available endomicroscopy technologies. PMID:18065276

  20. Combining multiphoton and CARS microscopy for skin imaging

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Weinigel, M.; Kellner-Höfer, M.; Bückle, R.; Darvin, M. E.; Lademann, J.; König, K.

    2013-02-01

    Microscopic imaging based on multiphoton fluorescence, second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) imaging has been realized in one common platform which is appropriate for use in hospitals. The different optical modalities non-invasively provide in vivo images from human skin with subcellular resolution, at different depths based on endogenous fluorescent, SHG-active molecules as well as non-fluorescent molecules with vibrational resonances at 2845 cm-1, in particular lipids. An overview of the system employing a Ti:sapphire laser and photonic crystal fiber to generate the excitation light as well as several imaging examples are presented.

  1. Intravital multiphoton imaging of mouse tibialis anterior muscle

    PubMed Central

    Lau, Jasmine; Goh, Chi Ching; Devi, Sapna; Keeble, Jo; See, Peter; Ginhoux, Florent; Ng, Lai Guan

    2016-01-01

    ABSTRACT Intravital imaging by multiphoton microscopy is a powerful tool to gain invaluable insight into tissue biology and function. Here, we provide a step-by-step tissue preparation protocol for imaging the mouse tibialis anterior skeletal muscle. Additionally, we include steps for jugular vein catheterization that allow for well-controlled intravenous reagent delivery. Preparation of the tibialis anterior muscle is minimally invasive, reducing the chances of inducing damage and inflammation prior to imaging. The tibialis anterior muscle is useful for imaging leukocyte interaction with vascular endothelium, and to understand muscle contraction biology. Importantly, this model can be easily adapted to study neuromuscular diseases and myopathies. PMID:28243520

  2. Microstructure imaging of human rectal mucosa using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liu, N. R.; Chen, G.; Chen, J. X.; Yan, J.; Zhuo, S. M.; Zheng, L. Q.; Jiang, X. S.

    2011-01-01

    Multiphoton microscopy (MPM) has high resolution and sensitivity. In this study, MPM was used to image microstructure of human rectal mucosa. The morphology and distribution of the main components in mucosa layer, absorptive cells and goblet cells in the epithelium, abundant intestinal glands in the lamina propria and smooth muscle fibers in the muscularis mucosa were clearly monitored. The variations of these components were tightly relevant to the pathology in gastrointestine system, especially early rectal cancer. The obtained images will be helpful for the diagnosis of early colorectal cancer.

  3. Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus

    NASA Astrophysics Data System (ADS)

    Sun, Yen; Lo, Wen; Lin, Sung-Jan; Lin, Wei-Chou; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2006-02-01

    The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover, increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area. In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.

  4. In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

    PubMed

    Kantelhardt, Sven R; Kalasauskas, Darius; König, Karsten; Kim, Ella; Weinigel, Martin; Uchugonova, Aisada; Giese, Alf

    2016-05-01

    High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflex™ Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

  5. Multiphoton imaging of basal cell carcinoma (BCC)

    NASA Astrophysics Data System (ADS)

    Cicchi, R.; Carli, P.; Massi, D.; Sestini, S.; Stambouli, D.; Pavone, F. S.

    2006-02-01

    We used two-photon microscopy towards the imaging of cutaneous basal cell carcinoma (BCC). Our aim was to evaluate the morphology of BCC using two-photon fluorescence excitation and to establish a correlation with histopathology. We built a custom two-photon microscope and we measured the system capabilities. The system allowed to perform a preliminary measurement on a fresh human skin tissue sample. A human skin tissue sample of BCC excised during dermatological surgery procedures were used. The clinical diagnosis of BCC was confirmed by subsequent histopathological examination. The sample was imaged using endogenous tissue fluorescence within 2-3 hours from the excision with a two photon laser scanning fluorescence microscope. The acquired images allowed an obvious discrimination of the neoplastic areas toward normal tissue, based on morphological differences and aberrations of the intensity of the fluorescence signal. Our results showed that BCC tissue has a more homogeneous structure in comparison to normal tissue as well as a higher fluorescent response. The images obtained by two photon microscopy were further compared to the images acquired by an optical microscope after the conventional histopathological examination on one part of the respective sample. Our suggested method may represent a new diagnostic tool that improves the diagnostic accuracy of clinical examination alone, enabling the accurate discrimination of basal cell carcinoma from normal tissue.

  6. In vivo multiphoton imaging of bile duct ligation

    NASA Astrophysics Data System (ADS)

    Liu, Yuan; Li, Feng-Chieh; Chen, Hsiao-Chin; Chang, Po-shou; Yang, Shu-Mei; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2008-02-01

    Bile is the exocrine secretion of liver and synthesized by hepatocytes. It is drained into duodenum for the function of digestion or drained into gallbladder for of storage. Bile duct obstruction is a blockage in the tubes that carry bile to the gallbladder and small intestine. However, Bile duct ligation results in the changes of bile acids in serum, liver, urine, and feces1, 2. In this work, we demonstrate a novel technique to image this pathological condition by using a newly developed in vivo imaging system, which includes multiphoton microscopy and intravital hepatic imaging chamber. The images we acquired demonstrate the uptake, processing of 6-CFDA in hepatocytes and excretion of CF in the bile canaliculi. In addition to imaging, we can also measure kinetics of the green fluorescence intensity.

  7. Multiphoton imaging: a view to understanding sulfur mustard lesions

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert J. S.; Madren-Whalley, Janna S.

    2003-07-01

    It is well known that topical exposure to sulfur mustard (SM) produces persistent, incapacitating blisters of the skin. However, the primary lesions effecting epidermal-dermal separation and disabling of mechanisms for cutaneous repair remain uncertain. Immunofluorescent staining plus multiphoton imaging of human epidermal tissues and keratinocytes exposed to SM (400 μM x 5 min)have revealed that SM disrupts adhesion-complex molecules which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin-14 showed early, progressive, postexposure collapse of the K5/K14 cytoskeleton that resulted in ventral displacement of the nuclei beneath its collapsing filaments. This effectively corrupted the dynamic filament assemblies that link basal-cell nuclei to the extracellular matrix via α6β4-integrin and laminin-5. At 1 h postexposure, there was disruption in the surface organization of α6β4 integrins, associated displacement of laminin-5 anchoring sites and a concomitant loss of functional asymmetry. Accordingly, our multiphoton images are providing compelling evidence that SM induces prevesicating lesions that disrupt the receptor-ligand organization and cytoskeletal systems required for maintaining dermal-epidermal attachment, signal transduction, and polarized mobility.

  8. Molecule-specific darkfield and multiphoton imaging using gold nanocages

    NASA Astrophysics Data System (ADS)

    Powless, Amy J.; Jenkins, Samir V.; McKay, Mary Lee; Chen, Jingyi; Muldoon, Timothy J.

    2015-03-01

    Due to their robust optical properties, biological inertness, and readily adjustable surface chemistry, gold nanostructures have been demonstrated as contrast agents in a variety of biomedical imaging applications. One application is dynamic imaging of live cells using bioconjugated gold nanoparticles to monitor molecule trafficking mechanisms within cells; for instance, the regulatory pathway of epidermal growth factor receptor (EGFR) undergoing endocytosis. In this paper, we have demonstrated a method to track endocytosis of EGFR in MDA-MB-468 breast adenocarcinoma cells using bioconjugated gold nanocages (AuNCs) and multiphoton microscopy. Dynamic imaging was performed using a time series capture of 4 images every minute for one hour. Specific binding and internalization of the bioconjugated AuNCs was observed while the two control groups showed non-specific binding at fewer surface sites, leading to fewer bound AuNCs and no internalization.

  9. Multibeam multifocal multiphoton photon counting imaging in scattering media

    NASA Astrophysics Data System (ADS)

    Hoover, Erich E.

    Multiphoton microscopy is an invaluable technique for the neurological community, allowing for deep explorations within highly scattering tissues such as the brain. However, prior to this research multiphoton microscopy was limited in its ability to rapidly construct volumetric images deep within scattering specimens. This work establishes a technique that permits such exploration through the application of multiple beams separated in both space and time, where signal photons corresponding to those beams are demultiplexed through the use of a field programmable gate array. With this system a number of improvements are provided to research in scattering media, including the coveted ability to perform photon-counting imaging with multiple beams. The ability to perform these measurements with multiple beams permits unique quantitative measurements of fluorophores within living specimens, allowing new research into dynamic three-dimensional behavior occurring within the brain. Additionally, the ability to perform multimodal measurements without filtering allows for unique avenues of research where the harmonic generation is indistinguishable from the two-photon excited fluorescence. These improvements provide neuroscience researchers with a large assortment of technological tools that will permit them to perform numerous novel experiments within the brain and other highly-scattering specimens, which should one day lead to significant advances in our understanding of complex neuronal activity.

  10. Live-Animal Imaging of Renal Function by Multiphoton Microscopy

    PubMed Central

    Dunn, Kenneth W.; Sutton, Timothy A.; Sandoval, Ruben M.

    2015-01-01

    Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high-speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. PMID:23042524

  11. Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy

    NASA Astrophysics Data System (ADS)

    Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Gibson, Emily A.; Lei, Tim C.

    2013-03-01

    The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

  12. Multiphoton imaging for assessing renal disposition in acute kidney injury

    NASA Astrophysics Data System (ADS)

    Liu, Xin; Liang, Xiaowen; Wang, Haolu; Roberts, Darren M.; Roberts, Michael S.

    2016-11-01

    Estimation of renal function and drug renal disposition in acute kidney injury (AKI), is important for appropriate dosing of drugs and adjustment of therapeutic strategies, but is challenging due to fluctuations in kidney function. Multiphoton microscopy has been shown to be a useful tool in studying drug disposition in liver and can reflect dynamic changes of liver function. We extend this imaging technique to investigate glomerular filtration rate (GFR) and tubular transporter functional change in various animal models of AKI, which mimic a broad range of causes of AKI such as hypoxia (renal ischemia- reperfusion), therapeutic drugs (e.g. cisplatin), rhabdomyolysis (e.g. glycerol-induced) and sepsis (e.g. LPSinduced). The MPM images revealed acute injury of tubular cells as indicated by reduced autofluorescence and cellular vacuolation in AKI groups compared to control group. In control animal, systemically injected FITC-labelled inulin was rapidly cleared from glomerulus, while the clearance of FITC-inulin was significantly delayed in most of animals in AKI group, which may reflect the reduced GFR in AKI. Following intravenous injection, rhodamine 123, a fluorescent substrate of p-glycoprotein (one of tubular transporter), was excreted into urine in proximal tubule via p-glycoprotein; in response to AKI, rhodamine 123 was retained in tubular cells as revealed by slower decay of fluorescence intensity, indicating P-gp transporter dysfunction in AKI. Thus, real-time changes in GFR and transporter function can be imaged in rodent kidney with AKI using multiphoton excitation of exogenously injected fluorescent markers.

  13. Multiphoton gonioscopy to image the trabecular meshwork of porcine eyes

    NASA Astrophysics Data System (ADS)

    Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Gibson, Emily A.; Lei, Tim C.

    2013-03-01

    The aqueous outflow system (AOS), including the trabecular meshwork (TM), the collector channels (CC) and the Schlemm's canal (SC), regulates intraocular pressure (IOP) through the drainage of the aqueous humor (AH). Abnormal IOP elevation leads to increased pressure stress to retinal ganglion cells, resulting in cell loss that can ultimately lead to complete loss of eyesight. Therefore, development of imaging tools to detect abnormal structural and functional changes of the AOS is important in early diagnosis and prevention of glaucoma. Multiphoton microscopy (MPM), including twophoton autofluorescence (TPAF) and second harmonic generation (SHG), is a label-free microscopic technique that allows molecular specific imaging of biological tissues like the TM. Since the TM and other AOS structures are located behind the highly scattering scleral tissue, transscleral imaging of the TM does not provide enough optical resolution. In this work, a gonioscopic lens is used to allow direct optical access of the TM through the cornea for MPM imaging. Compared to transscleral imaging, the acquired MPM images show improved resolution as individual collagen fiber bundles of the TM can be observed. MPM gonioscopy may have the potential to be developed as a future clinical imaging tool for glaucoma diagnostics.

  14. Superresolved multiphoton microscopy with spatial frequency-modulated imaging

    SciTech Connect

    Field, Jeffrey J.; Wernsing, Keith A.; Domingue, Scott R.; Allende Motz, Alyssa M.; DeLuca, Keith F.; Levi, Dean H.; DeLuca, Jennifer G.; Young, Michael D.; Squier, Jeff A.; Bartels, Randy A.

    2016-05-26

    Superresolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection. We show multimodal superresolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2..eta.. below the diffraction limit, where ..eta.. is the highest power of the nonlinear intensity response. MP-SPIFI can be used to provide enhanced resolution in optically thin media and may provide a solution for superresolved imaging deep in scattering media.

  15. Superresolved multiphoton microscopy with spatial frequency-modulated imaging

    PubMed Central

    Field, Jeffrey J.; Wernsing, Keith A.; Domingue, Scott R.; Allende Motz, Alyssa M.; DeLuca, Keith F.; Levi, Dean H.; DeLuca, Jennifer G.; Young, Michael D.; Squier, Jeff A.; Bartels, Randy A.

    2016-01-01

    Superresolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection. We show multimodal superresolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2η below the diffraction limit, where η is the highest power of the nonlinear intensity response. MP-SPIFI can be used to provide enhanced resolution in optically thin media and may provide a solution for superresolved imaging deep in scattering media. PMID:27231219

  16. In vivo multiphoton imaging of immune cell dynamics.

    PubMed

    Okada, Takaharu; Takahashi, Sonoko; Ishida, Azusa; Ishigame, Harumichi

    2016-11-01

    Multiphoton imaging has been utilized to analyze in vivo immune cell dynamics over the last 15 years. Particularly, it has deepened the understanding of how immune responses are organized by immune cell migration and interactions. In this review, we first describe the following technical advances in recent imaging studies that contributed to the new findings on the regulation of immune responses and inflammation. Improved multicolor imaging of immune cell behavior has revealed that their interactions are spatiotemporally coordinated to achieve efficient and long-term immunity. The use of photoactivatable and photoconvertible fluorescent proteins has increased duration and volume of cell tracking, even enabling the analysis of inter-organ migration of immune cells. In addition, visualization of immune cell activation using biosensors for intracellular calcium concentration and signaling molecule activities has started to give further mechanistic insights. Then, we also introduce recent imaging analyses of interactions between immune cells and non-immune cells including endothelial, fibroblastic, epithelial, and nerve cells. It is argued that future imaging studies that apply updated technical advances to analyze interactions between immune cells and non-immune cells will be important for thorough physiological understanding of the immune system.

  17. Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo

    NASA Astrophysics Data System (ADS)

    Larson, Daniel R.; Zipfel, Warren R.; Williams, Rebecca M.; Clark, Stephen W.; Bruchez, Marcel P.; Wise, Frank W.; Webb, Watt W.

    2003-05-01

    The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

  18. Multiphoton imaging with a novel compact diode-pumped Ti:sapphire oscillator.

    PubMed

    König, Karsten; Andersen, Peter; Le, Tuan; Breunig, Hans Georg

    2015-12-01

    Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal-footprint Ti:sapphire oscillator, pumped by a frequency-doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its imaging capability using different biological samples, i.e. cell monolayers, corneal tissue, and human skin. With the novel laser, the realization of very compact Ti:sapphire-based systems for high-quality multiphoton imaging at a significantly size and weight compared to current systems will become possible.

  19. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.

    PubMed

    Larson, Daniel R; Zipfel, Warren R; Williams, Rebecca M; Clark, Stephen W; Bruchez, Marcel P; Wise, Frank W; Webb, Watt W

    2003-05-30

    The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

  20. Multiphoton imaging the disruptive nature of sulfur mustard lesions

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert J.; Braue, Catherine R.; Dillman, James F.

    2005-03-01

    Sulfur mustard [bis-2-chloroethyl sulfide] is a vesicating agent first used as a weapon of war in WWI. It causes debilitating blisters at the epidermal-dermal junction and involves molecules that are also disrupted by junctional epidermolysis bullosa (JEB) and other blistering skin diseases. Despite its recurring use in global conflicts, there is still no completely effective treatment. We have shown by imaging human keratinocytes in cell culture and in intact epidermal tissues that the basal cells of skin contain well-organized molecules (keratins K5/K14, α6β4 integrin, laminin 5 and α3β1 integrin) that are early targets of sulfur mustard. Disruption and collapse of these molecules is coincident with nuclear displacement, loss of functional asymmetry, and loss of polarized mobility. The progression of this pathology precedes basal cell detachment by 8-24 h, a time equivalent to the "clinical latent phase" that defines the extant period between agent exposure and vesication. Our images indicate that disruption of adhesion-complex molecules also impairs cytoskeletal proteins and the integration of structures required for signal transduction and tissue repair. We have recently developed an optical system to test this hypothesis, i.e., to determine whether and how the early disruption of target molecules alters signal transduction. This environmentally controlled on-line system provides a nexus for real-time correlation of imaged lesions with DNA microarray analysis, and for using multiphoton microscopy to facilitate development of more effective treatment strategies.

  1. Multiphoton imaging of low grade, high grade intraepithelial neoplasia and intramucosal invasive cancer of esophagus

    NASA Astrophysics Data System (ADS)

    Xu, Jian; Jiang, Liwei; Kang, Deyong; Wu, Xuejing; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Lin, Jiangbo; Chen, Jianxin

    2017-04-01

    Esophageal squamous cell carcinoma (ESCC) is devastating because of its aggressive lymphatic spread and clinical course. It is believed to occur through low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), and intramucosal invasive cancer (IMC) before transforming to submucosal cancer. In particular, these early lesions (LGIN, HGIN and IMC), which involve no lymph node nor distant metastasis, can be cured by endoscopic treatment. Therefore, early identification of these lesions is important so as to offer a curative endoscopic resection, thus slowing down the development of ESCC. In this work, spectral information and morphological features of the normal esophageal mucosa are first studied. Then, the morphological changes of LGIN, HGIN and IMC are described. Lastly, quantitative parameters are also extracted by calculating the nuclear-to-cytoplasmic ratio of epithelial cells and the pixel density of collagen in the lamina propria. These results show that multiphoton microscopy (MPM) has the ability to identify normal esophageal mucosa, LGIN, HGIN and IMC. With the development of multiphoton endoscope systems for in vivo imaging, combined with a laser ablation system, MPM has the potential to provide immediate pathologic diagnosis and curative treatment of ESCC before the transformation to submucosal cancer in the future.

  2. In vivo multiphoton microscopy associated to 3D image processing for human skin characterization

    NASA Astrophysics Data System (ADS)

    Baldeweck, T.; Tancrède, E.; Dokladal, P.; Koudoro, S.; Morard, V.; Meyer, F.; Decencière, E.; Pena, A.-M.

    2012-03-01

    Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspectR medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

  3. Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

    PubMed Central

    Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

    2009-01-01

    We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

  4. Invited review article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy.

    PubMed

    Carriles, Ramón; Schafer, Dawn N; Sheetz, Kraig E; Field, Jeffrey J; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W; Squier, Jeffrey A

    2009-08-01

    We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.

  5. Intravital Multiphoton Imaging of the Kidney: Tubular Structure and Metabolism.

    PubMed

    Small, David M; Sanchez, Washington Y; Gobe, Glenda C

    2016-01-01

    Multiphoton microscopy (MPM) allows the visualization of dynamic pathophysiological events in real time in live animals. Intravital imaging can be applied to investigate novel mechanisms and treatments of different forms of kidney disease as well as improve our understanding of normal kidney physiology. Using rodent models, in conjunction with endogenous fluorescence and infused exogenous fluorescent dyes, measurement can be made of renal processes such as glomerular permeability, juxtaglomerular apparatus function, interactions of the tubulointerstitium, tubulovascular interactions, vascular flow rate, and the renin-angiotensin-aldosterone system. Subcellular processes including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes of apoptosis and necrosis can also be seen and measured by MPM. The current methods chapter presents an overview of MPM with a focus on techniques for intravital kidney imaging and gives examples of instances where intravital MPM has been utilized to study renal pathophysiology. Suggestions are provided for MPM methods within the confines of intravital microscopy and selected kidney structure. MPM is undoubtedly a powerful new technique for application in experimental nephrology, and we believe it will continue to create new paradigms for understanding and treating kidney disease.

  6. Optimal spectral filtering in soliton self-frequency shift for deep-tissue multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Ke; Qiu, Ping

    2015-05-01

    Tunable optical solitons generated by soliton self-frequency shift (SSFS) have become valuable tools for multiphoton microscopy (MPM). Recent progress in MPM using 1700 nm excitation enabled visualizing subcortical structures in mouse brain in vivo for the first time. Such an excitation source can be readily obtained by SSFS in a large effective-mode-area photonic crystal rod with a 1550-nm fiber femtosecond laser. A longpass filter was typically used to isolate the soliton from the residual in order to avoid excessive energy deposit on the sample, which ultimately leads to optical damage. However, since the soliton was not cleanly separated from the residual, the criterion for choosing the optimal filtering wavelength is lacking. Here, we propose maximizing the ratio between the multiphoton signal and the n'th power of the excitation pulse energy as a criterion for optimal spectral filtering in SSFS when the soliton shows dramatic overlapping with the residual. This optimization is based on the most efficient signal generation and entirely depends on physical quantities that can be easily measured experimentally. Its application to MPM may reduce tissue damage, while maintaining high signal levels for efficient deep penetration.

  7. Optimal spectral filtering in soliton self-frequency shift for deep-tissue multiphoton microscopy.

    PubMed

    Wang, Ke; Qiu, Ping

    2015-05-01

    Tunable optical solitons generated by soliton self-frequency shift (SSFS) have become valuable tools for multiphoton microscopy (MPM). Recent progress in MPM using 1700 nm excitation enabled visualizing subcortical structures in mouse brain in vivo for the first time. Such an excitation source can be readily obtained by SSFS in a large effective-mode-area photonic crystal rod with a 1550-nm fiber femtosecond laser. A longpass filter was typically used to isolate the soliton from the residual in order to avoid excessive energy deposit on the sample, which ultimately leads to optical damage. However, since the soliton was not cleanly separated from the residual, the criterion for choosing the optimal filtering wavelength is lacking. Here, we propose maximizing the ratio between the multiphoton signal and the n'th power of the excitation pulse energy as a criterion for optimal spectral filtering in SSFS when the soliton shows dramatic overlapping with the residual. This optimization is based on the most efficient signal generation and entirely depends on physical quantities that can be easily measured experimentally. Its application to MPM may reduce tissue damage, while maintaining high signal levels for efficient deep penetration.

  8. High-throughput multiphoton-induced three-dimensional ablation and imaging for biotissues.

    PubMed

    Lin, Chun-Yu; Li, Pei-Kao; Cheng, Li-Chung; Li, Yi-Cheng; Chang, Chia-Yuan; Chiang, Ann-Shyn; Dong, Chen Yuan; Chen, Shean-Jen

    2015-02-01

    In this study, a temporal focusing-based high-throughput multiphoton-induced ablation system with axially-resolved widefield multiphoton excitation has been successfully applied to rapidly disrupt biotissues. Experimental results demonstrate that this technique features high efficiency for achieving large-area laser ablation without causing serious photothermal damage in non-ablated regions. Furthermore, the rate of tissue processing can reach around 1.6 × 10(6) μm(3)/s in chicken tendon. Moreover, the temporal focusing-based multiphoton system can be efficiently utilized in optical imaging through iterating high-throughput multiphoton-induced ablation machining followed by widefield optical sectioning; hence, it has the potential to obtain molecular images for a whole bio-specimen.

  9. High-throughput multiphoton-induced three-dimensional ablation and imaging for biotissues

    PubMed Central

    Lin, Chun-Yu; Li, Pei-Kao; Cheng, Li-Chung; Li, Yi-Cheng; Chang, Chia-Yuan; Chiang, Ann-Shyn; Dong, Chen Yuan; Chen, Shean-Jen

    2015-01-01

    In this study, a temporal focusing-based high-throughput multiphoton-induced ablation system with axially-resolved widefield multiphoton excitation has been successfully applied to rapidly disrupt biotissues. Experimental results demonstrate that this technique features high efficiency for achieving large-area laser ablation without causing serious photothermal damage in non-ablated regions. Furthermore, the rate of tissue processing can reach around 1.6 × 106 μm3/s in chicken tendon. Moreover, the temporal focusing-based multiphoton system can be efficiently utilized in optical imaging through iterating high-throughput multiphoton-induced ablation machining followed by widefield optical sectioning; hence, it has the potential to obtain molecular images for a whole bio-specimen. PMID:25780739

  10. Infrared transform spectral imager

    NASA Astrophysics Data System (ADS)

    Vujkovic-Cvijin, Pajo; Lee, Jamine; Gregor, Brian; Goldstein, Neil; Panfili, Raphael; Fox, Marsha

    2012-10-01

    A dispersive transform spectral imager named FAROS (FAst Reconfigurable Optical Sensor) has been developed for high frame rate, moderate-to-high resolution hyperspectral imaging. A programmable digital micromirror array (DMA) modulator makes it possible to adjust spectral, temporal and spatial resolution in real time to achieve optimum tradeoff for dynamic monitoring requirements. The system's F/2.8 collection optics produces diffraction-limited images in the mid-wave infrared (MWIR) spectral region. The optical system is based on a proprietary dual-pass Offner configuration with a single spherical mirror and a confocal spherical diffraction grating. FAROS fulfills two functions simultaneously: one output produces two-dimensional polychromatic imagery at the full focal plane array (FPA) frame rate for fast object acquisition and tracking, while the other output operates in parallel and produces variable-resolution spectral images via Hadamard transform encoding to assist in object discrimination and classification. The current version of the FAROS spectral imager is a multispectral technology demonstrator that operates in the MWIR with a 320 x 256 pixel InSb FPA running at 478 frames per second resulting in time resolution of several tens of milliseconds per hypercube. The instrument has been tested by monitoring small-scale rocket engine firings in outdoor environments. The instrument has no macro-scale moving parts, and conforms to a robust, small-volume and lightweight package, suitable for integration with small surveillance vehicles. The technology is also applicable to multispectral/hyperspectral imaging applications in diverse areas such as atmospheric contamination monitoring, agriculture, process control, and biomedical imaging, and can be adapted for use in any spectral domain from the ultraviolet (UV) to the LWIR region.

  11. Three-dimensional tooth imaging using multiphoton and second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Min-Huey; Chen, Wei-Liang; Sun, Yen; Fwu, Peter Tramyeon; Lin, Ming-Gu; Dong, Chen-Yuan

    2007-02-01

    Detailed morphological and cellular information relating to the human tooth have traditionally been obtained through histological studies that required decalcification, staining, and fixation. With the recent invention of multiphoton microscopy, it has become possible to acquire high resolution images without histological procedures. Using an epiilluminated multiphoton microscope, we obtained two-photon excited autofluorescence and second harmonic generation (SHG) images of ex vivo human tooth. By combining these two imaging modalities we obtained submicron resolution images of the enamel, dentin, and the periodontal ligaments. The enamel emits endogenous two-photon autofluorescence. The structure of the dentin is visible from both the autofluorescence and second harmonic generation signals. The periodontal ligament composed mostly of collagen can be visualized by SHG imaging. We also constructed three dimensional images of the enamel, dentin, and periodontal ligament. The effectiveness of using multiphoton and second harmonic generation microscopy to obtain structural information of teeth suggest its potential use in dental diagnostics.

  12. Multi Spectral Imaging System

    NASA Technical Reports Server (NTRS)

    Spiering, Bruce A. (Inventor)

    1999-01-01

    An optical imaging system provides automatic co-registration of a plurality of multi spectral images of an object which are generated by a plurality of video cameras or other optical detectors. The imaging system includes a modular assembly of beam splitters, lens tubes, camera lenses and wavelength selective filters which facilitate easy reconfiguration and adjustment of the system for various applications. A primary lens assembly generates a real image of an object to be imaged on a reticle which is positioned at a fixed length from a beam splitter assembly. The beam splitter assembly separates a collimated image beam received from the reticle into multiple image beams, each of which is projected onto a corresponding one of a plurality of video cameras. The lens tubes which connect the beam splitter assembly to the cameras are adjustable in length to provide automatic co-registration of the images generated by each camera.

  13. Multipurpose Spectral Imager

    NASA Astrophysics Data System (ADS)

    Sigernes, Fred; Lorentzen, Dag Arne; Heia, Karsten; Svenøe, Trond

    2000-06-01

    A small spectral imaging system is presented that images static or moving objects simultaneously as a function of wavelength. The main physical principle is outlined and demonstrated. The instrument is capable of resolving both spectral and spatial information from targets throughout the entire visible region. The spectral domain has a bandpass of 12 . One can achieve the spatial domain by rotating the system s front mirror with a high-resolution stepper motor. The spatial resolution range from millimeters to several meters depends mainly on the front optics used and whether the target is fixed (static) or movable relative to the instrument. Different applications and examples are explored, including outdoor landscapes, industrial fish-related targets, and ground-level objects observed in the more traditional way from an airborne carrier (remote sensing). Through the examples, we found that the instrument correctly classifies whether a shrimp is peeled and whether it can disclose the spectral and spatial microcharacteristics of targets such as a fish nematode (parasite). In the macroregime, we were able to distinguish a marine vessel from the surrounding sea and sky. A study of the directional spectral albedo from clouds, mountains, snow cover, and vegetation has also been included. With the airborne experiment, the imager successfully classified snow cover, leads, and new and rafted ice, as seen from 10.000 ft (3.048 m).

  14. Two-photon imaging of intact living plants during freezing with a flexible multiphoton tomograph

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; König, K.

    2015-02-01

    We describe the combination of a flexible multiphoton tomograph (MPTflex) with a heating and cooling stage. The stage allows temperature control in the range of (-196 °C) (77 K) to +600 °C (873 K) with selectable heating/freezing rates between 0.01 K min-1 and 150 K min-1. To illustrate the imaging capabilities of the combined system, fluorescence intensity and lifetime of intrinsic molecules from a plant leaf were imaged with submicron resolution during freezing in vivo without detaching the leaf from the plant. An increase of fluorescence intensity and decay times with decreasing temperature was observed. The measurements illustrate the usefulness of multiphoton imaging as a non-invasive online tool to investigate temperature-induced effects. The flexible multiphoton tomograph with its adjustable mechano-optical arm and scan head allows imaging at otherwise hardly accessible sample regions.

  15. Rapid diagnosis of liver fibrosis using multimodal multiphoton nonlinear optical microspectroscopy imaging.

    PubMed

    Lee, Jang Hyuk; Kim, Jong Chul; Tae, Giyoong; Oh, Myoung-Kyu; Ko, Do-Kyeong

    2013-07-01

    A multimodal multiphoton nonlinear optical (NLO) microspectroscopy imaging system was developed using a femtosecond laser and a photonic crystal fiber. Coherent anti-Stokes Raman scattering (CARS) microspectroscopy was combined with two-photon excitation fluorescence and second-harmonic generation microscopy in one platform and the system was applied to diagnose liver fibrosis. Normal and liver fibrosis tissues were clearly distinguished with the great difference from CARS spectra as well as multimodal multiphoton NLO images. We expect the system to be a rapid diagnosis tool for liver fibrosis at tissue level with label-free imaging of significant biochemical components.

  16. Multiphoton fluorescent images with a spatially varying background signal: a ML deconvolution method.

    PubMed

    Crivaro, M; Enjieu-Kadji, H; Hatanaka, R; Nakauchi, S; Bosch, J; Judin, J; Riera, J; Kawashima, R

    2011-06-01

    By means of multiphoton laser scanning microscopy, neuroscientists can look inside the brain deeper than has ever been possible before. Multiphoton fluorescent images, as all optical images, suffer from degradation caused by a variety of sources (e.g. light dispersion and absorption in the tissue, laser fluctuations, spurious photodetection and staining deficiency). From a modelling perspective, such degradations can be considered the sum of stochastic noise and a background signal. Among the methods proposed in the literature to perform image deconvolution in either confocal or multiphoton fluorescent microscopy, Vicidomini et al. (2009) were the first to incorporate models for noise (a Poisson process) and background signal (spatially constant) in the context of regularized inverse problems. Unfortunately, the so-called split-gradient deconvolution method (SGM) they used did not consider possible spatial variations in the background signal. In this paper, we extend the SGM by adding a maximum-likelihood estimation step for the determination of a spatially varying background signal. We demonstrate that the assumption of a constant background is not always valid in multiphoton laser microscopy and by using synthetic and actual multiphoton fluorescent images, we evaluate the face of validity of the proposed method, and compare its accuracy with the previously introduced SGM algorithm.

  17. Multiphoton Spectral Analysis of Benzo[a]pyrene Uptake and Metabolism in a Rat Liver Cell Line

    PubMed Central

    Barhoumi, Rola; Mouneimne, Youssef; Ramos, Ernesto; Morisseau, Christophe; Hammock, Bruce D.; Safe, Stephen; Parrish, Alan R.; Burghardt, Robert C

    2011-01-01

    Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an “advanced unmixing process”, identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 hr. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis. PMID:21420996

  18. Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in a rat liver cell line

    SciTech Connect

    Barhoumi, Rola; Mouneimne, Youssef; Ramos, Ernesto; Morisseau, Christophe; Hammock, Bruce D.; Safe, Stephen; Parrish, Alan R.; Burghardt, Robert C.

    2011-05-15

    Dynamic analysis of the uptake and metabolism of polycyclic aromatic hydrocarbons (PAHs) and their metabolites within live cells in real time has the potential to provide novel insights into genotoxic and non-genotoxic mechanisms of cellular injury caused by PAHs. The present work, combining the use of metabolite spectra generated from metabolite standards using multiphoton spectral analysis and an 'advanced unmixing process', identifies and quantifies the uptake, partitioning, and metabolite formation of one of the most important PAHs (benzo[a]pyrene, BaP) in viable cultured rat liver cells over a period of 24 h. The application of the advanced unmixing process resulted in the simultaneous identification of 8 metabolites in live cells at any single time. The accuracy of this unmixing process was verified using specific microsomal epoxide hydrolase inhibitors, glucuronidation and sulfation inhibitors as well as several mixtures of metabolite standards. Our findings prove that the two-photon microscopy imaging surpasses the conventional fluorescence imaging techniques and the unmixing process is a mathematical technique that seems applicable to the analysis of BaP metabolites in living cells especially for analysis of changes of the ultimate carcinogen benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide. Therefore, the combination of the two-photon acquisition with the unmixing process should provide important insights into the cellular and molecular mechanisms by which BaP and other PAHs alter cellular homeostasis.

  19. Application of ultrafast gold luminescence to measuring the instrument response function for multispectral multiphoton fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Talbot, Clifford B.; Patalay, Rakesh; Munro, Ian; Warren, Sean; Ratto, Fulvio; Matteini, Paolo; Pini, Roberto; Breunig, H. Georg; König, Karsten; Chu, Antony C.; Stamp, Gordon W.; Neil, Mark A. A.; French, Paul M. W.; Dunsby, Chris

    2011-07-01

    When performing multiphoton fluorescence lifetime imaging in multiple spectral emission channels, an instrument response function must be acquired in each channel if accurate measurements of complex fluorescence decays are to be performed. Although this can be achieved using the reference reconvolution technique, it is difficult to identify suitable fluorophores with a mono-exponential fluorescence decay across a broad emission spectrum. We present a solution to this problem by measuring the IRF using the ultrafast luminescence from gold nanorods. We show that ultrafast gold nanorod luminescence allows the IRF to be directly obtained in multiple spectral channels simultaneously across a wide spectral range. We validate this approach by presenting an analysis of multispectral autofluorescence FLIM data obtained from human skin ex vivo.

  20. Real-time optical diagnosis of gastric cancer with serosal invasion using multiphoton imaging

    PubMed Central

    Yan, Jun; Zheng, Yu; Zheng, Xiaoling; Liu, Zhangyuanzhu; Liu, Wenju; Chen, Dexin; Dong, Xiaoyu; Li, Kai; Liu, Xiumin; Chen, Gang; Lu, Jianping; Chen, Jianxin; Zhuo, Shuangmu; Li, Guoxin

    2016-01-01

    A real-time optical biopsy, which could determine tissue histopathology, would be of extraordinary benefit to staging laparoscopy for gastric cancer with serosal invasion (T4) that requires downstage treatment. We investigated the feasibility of using multiphoton imaging to perform a real-time optical diagnosis of gastric cancer with or without serosal invasion. First, a pilot study was performed to establish the optical diagnostic features of gastric cancer with or without serosal invasion using multiphoton imaging compared with hematoxylin-eosin staining and Masson’s trichrome staining. Second, a blinded study was performed to compare the diagnostic sensitivity, specificity, and accuracy of multiphoton imaging and endoscopic ultrasonography (EUS) for T4 gastric cancer. In the pilot study, multiphoton imaging revealed collagen loss and degradation and cellular and nuclear pleomorphism in gastric cancer with serosal invasion. The collagen content in gastric cancer with or without serosal invasion was 0.36 ± 0.18 and 0.79 ± 0.16 (p < 0.001), respectively. In the blinded study, the sensitivity, specificity, and accuracy of EUS and multiphoton imaging for T4 gastric cancer were 70% and 90% (p = 0.029), 66.67% and 96.67% (p = 0.003), and 68.33% and 93.33% (p = 0.001), respectively. It is feasible to use multiphoton imaging to make a real-time optical diagnosis of gastric cancer with or without serosal invasion. PMID:27499365

  1. Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration.

    PubMed

    Bueno, Juan M; Skorsetz, Martin; Palacios, Raquel; Gualda, Emilio J; Artal, Pablo

    2014-01-01

    Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.

  2. Label-free multiphoton imaging and photoablation of preinvasive cancer cells

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Wu, Guizhu; Zhu, Xiaoqin; Jiang, Xingshan; Xie, Shusen

    2012-01-01

    Detection and treatment of early lesions in epithelial tissue offer several possibilities for curing cancer, but it is challenging. Here, we present an optical technique, the combination of multiphoton imaging and absorption, to label-freely detect and ablate preinvasive cancer cells in epithelial tissue. We find that multiphoton imaging can label-freely visualize the principal features of nuclear atypia associated with epithelial precancerous lesions, and the spatial localization of multiphoton absorption can perform targeted ablation of preinvasive cancer cells with micrometer-sized volume precision. These results indicate that this optical technique has the capability to label-freely visualize and remove preinvasive cancer cells in epithelial tissue. This study highlights the potential of this technique as a "seek-and-treat" tool for early lesions in epithelial tissue.

  3. Differentiation of normal and cancerous lung tissues by multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Wu, Ruei-Jhih; Hovhannisyan, Vladimir A.; Lin, Wei-Chou; Lin, Sung-Jan; So, Peter T. C.; Dong, Chen-Yuan

    2009-07-01

    We utilize multiphoton microscopy for the label-free diagnosis of noncancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from humans. Our results show that the combination of second-harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from noncancerous lung tissues. Specifically, noncancerous lung tissues are largely fibrotic in structure, while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55+/-0.23 and 0.87+/-0.15, respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50+/-0.12 and 0.72+/-0.13, respectively. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from noncancerous tissues.

  4. Differentiation of normal and cancerous lung tissues by multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Wu, Ruei-Jr; Hovhannisyan, Vladimir A.; Lin, Wei-Chou; Lin, Sung-Jan; So, Peter T. C.; Dong, Chen-Yuan

    2010-02-01

    In this work, we utilized multiphoton microscopy for the label-free diagnosis of non-cancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from human. Our results show that the combination of second harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from non-cancerous lung tissues. Specifically, non-cancerous lung tissues are largely fibrotic in structure while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI or SAAID) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55 +/-0.23 and 0.87+/-0.15 respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50+/-0.12 and 0.72+/-0.13 respectively. Intrinsic fluorescence ratio (FAD/NADH) of SCC and non-cancerous tissues are 0.40+/-0.05 and 0.53+/-0.05 respectively, the redox ratio of SCC diminishes significantly, indicating that increased cellular metabolic activity. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from non-cancerous tissues. With additional development, multiphoton microscopy may be used for the clinical diagnosis of lung cancers.

  5. Clinical combination of multiphoton tomography and high frequency ultrasound imaging for evaluation of skin diseases

    NASA Astrophysics Data System (ADS)

    König, K.; Speicher, M.; Koehler, M. J.; Scharenberg, R.; Elsner, P.; Kaatz, M.

    2010-02-01

    For the first time, high frequency ultrasound imaging, multiphoton tomography, and dermoscopy were combined in a clinical study. Different dermatoses such as benign and malign skin cancers, connective tissue diseases, inflammatory skin diseases and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art and highly sophisticated ultrasound systems for dermatology, (ii) the femtosecond-laser multiphoton tomograph DermaInspectTM and (iii) dermoscopes. Dermoscopy provides two-dimensional color imaging of the skin surface with a magnification up to 70x. Ultrasound images are generated from reflections of the emitted ultrasound signal, based on inhomogeneities of the tissue. These echoes are converted to electrical signals. Depending on the ultrasound frequency the penetration depth varies from about 1 mm to 16 mm in dermatological application. The 100-MHz-ultrasound system provided an axial resolution down to 16 μm and a lateral resolution down to 32 μm. In contrast to the wide-field ultrasound images, multiphoton tomography provided horizontal optical sections of 0.36×0.36 mm2 down to 200 μm tissue depth with submicron resolution. The autofluorescence of mitochondrial coenzymes, melanin, and elastin as well as the secondharmonic- generation signal of the collagen network were imaged. The combination of ultrasound and multiphoton tomography provides a novel opportunity for diagnostics of skin disorders.

  6. Quantitative determination of maximal imaging depth in all-NIR multiphoton microscopy images of thick tissues

    NASA Astrophysics Data System (ADS)

    Sarder, Pinaki; Akers, Walter J.; Sudlow, Gail P.; Yazdanfar, Siavash; Achilefu, Samuel

    2014-02-01

    We report two methods for quantitatively determining maximal imaging depth from thick tissue images captured using all-near-infrared (NIR) multiphoton microscopy (MPM). All-NIR MPM is performed using 1550 nm laser excitation with NIR detection. This method enables imaging more than five-fold deep in thick tissues in comparison with other NIR excitation microscopy methods. In this study, we show a correlation between the multiphoton signal along the depth of tissue samples and the shape of the corresponding empirical probability density function (pdf) of the photon counts. Histograms from this analysis become increasingly symmetric with the imaging depth. This distribution transitions toward the background distribution at higher imaging depths. Inspired by these observations, we propose two independent methods based on which one can automatically determine maximal imaging depth in the all-NIR MPM images of thick tissues. At this point, the signal strength is expected to be weak and similar to the background. The first method suggests the maximal imaging depth corresponds to the deepest image plane where the ratio between the mean and median of the empirical photon-count pdf is outside the vicinity of 1. The second method suggests the maximal imaging depth corresponds to the deepest image plane where the squared distance between the empirical photon-count mean obtained from the object and the mean obtained from the background is greater than a threshold. We demonstrate the application of these methods in all-NIR MPM images of mouse kidney tissues to study maximal depth penetration in such tissues.

  7. Clinical multiphoton FLIM tomography

    NASA Astrophysics Data System (ADS)

    König, Karsten

    2012-03-01

    This paper gives an overview on current clinical high resolution multiphoton fluorescence lifetime imaging in volunteers and patients. Fluorescence lifetime imaging (FLIM) in Life Sciences was introduced in Jena/Germany in 1988/89 based on a ZEISS confocal picosecond dye laser scanning microscope equipped with a single photon counting unit. The porphyrin distribution in living cells and living tumor-bearing mice was studied with high spatial, temporal, and spectral resolution. Ten years later, time-gated cameras were employed to detect dental caries in volunteers based on one-photon excitation of autofluorescent bacteria with long fluorescence lifetimes. Nowadays, one-photon FLIM based on picosecond VIS laser diodes are used to study ocular diseases in humans. Already one decade ago, first clinical twophoton FLIM images in humans were taken with the certified clinical multiphoton femtosecond laser tomograph DermaInspectTM. Multiphoton tomographs with FLIM modules are now operating in hospitals at Brisbane, Tokyo, Berlin, Paris, London, Modena and other European cities. Multiple FLIM detectors allow spectral FLIM with a temporal resolution down to 20 ps (MCP) / 250 ps (PMT) and a spectral resolution of 10 nm. Major FLIM applications include the detection of intradermal sunscreen and tattoo nanoparticles, the detection of different melanin types, the early diagnosis of dermatitis and malignant melanoma, as well as the measurement of therapeutic effects in pateints suffering from dermatitis. So far, more than 1,000 patients and volunteers have been investigated with the clinical multiphoton FLIM tomographs DermaInspectTM and MPTflexTM.

  8. A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

    PubMed Central

    Poland, Simon P.; Krstajić, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

    2015-01-01

    We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction. PMID:25780724

  9. Label-free imaging and quantitative chemical analysis of Alzheimer's disease brain samples with multimodal multiphoton nonlinear optical microspectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, Jang Hyuk; Kim, Dae Hwan; Song, Woo Keun; Oh, Myoung-Kyu; Ko, Do-Kyeong

    2015-05-01

    We developed multimodal multiphoton microspectroscopy using a small-diameter probe with gradient-index lenses and applied it to unstained Alzheimer's disease (AD) brain samples. Our system maintained the image quality and spatial resolution of images obtained using an objective lens of similar numerical aperture. Multicolor images of AD brain samples were obtained simultaneously by integrating two-photon excited fluorescence and second-harmonic generation on a coherent anti-Stokes Raman scattering (CARS) microendoscope platform. Measurements of two hippocampal regions, the cornus ammonis-1 and dentate gyrus, revealed more lipids, amyloid fibers, and collagen in the AD samples than in the normal samples. Normal and AD brains were clearly distinguished by a large spectral difference and quantitative analysis of the CH mode using CARS microendoscope spectroscopy. We expect this system to be an important diagnosis tool in AD research.

  10. Label-free imaging and quantitative chemical analysis of Alzheimer's disease brain samples with multimodal multiphoton nonlinear optical microspectroscopy.

    PubMed

    Lee, Jang Hyuk; Kim, Dae Hwan; Song, Woo Keun; Oh, Myoung-Kyu; Ko, Do-Kyeong

    2015-05-01

    We developed multimodal multiphoton microspectroscopy using a small-diameter probe with gradient-index lenses and applied it to unstained Alzheimer's disease (AD) brain samples. Our system maintained the image quality and spatial resolution of images obtained using an objective lens of similar numerical aperture. Multicolor images of AD brain samples were obtained simultaneously by integrating two-photon excited fluorescence and second-harmonic generation on a coherent anti-Stokes Raman scattering (CARS) microendoscope platform. Measurements of two hippocampal regions, the cornus ammonis-1 and dentate gyrus, revealed more lipids, amyloid fibers, and collagen in the AD samples than in the normal samples. Normal and AD brains were clearly distinguished by a large spectral difference and quantitative analysis of the CH mode using CARS microendoscope spectroscopy. We expect this system to be an important diagnosis tool in AD research

  11. Compact diode laser source for multiphoton biological imaging

    PubMed Central

    Niederriter, Robert D.; Ozbay, Baris N.; Futia, Gregory L.; Gibson, Emily A.; Gopinath, Juliet T.

    2016-01-01

    We demonstrate a compact, pulsed diode laser source suitable for multiphoton microscopy of biological samples. The center wavelength is 976 nm, near the peak of the two-photon cross section of common fluorescent markers such as genetically encoded green and yellow fluorescent proteins. The laser repetition rate is electrically tunable between 66.67 kHz and 10 MHz, with 2.3 ps pulse duration and peak powers >1 kW. The laser components are fiber-coupled and scalable to a compact package. We demonstrate >600 μm depth penetration in brain tissue, limited by laser power. PMID:28101420

  12. Coupling CARS with multiphoton fluorescence and high harmonic generation imaging modalities using a femtosecond laser source

    NASA Astrophysics Data System (ADS)

    Chen, Hongtao; Slipchenko, Mikhail N.; Zhu, Jiabin; Buhman, Kimberly K.; Cheng, Ji-Xin

    2009-02-01

    Multimodal nonlinear optical imaging has opened new opportunities and becomes a powerful tool for imaging complex tissue samples with inherent 3D spatial resolution.. We present a robust and easy-to-operate approach to add the coherent anti-stokes Raman scattering (CARS) imaging modality to a widely used multiphoton microscope. The laser source composed of a Mai Tai femtosecond laser and an optical parametric oscillator (OPO) offers one-beam, two-beam and three-beam modalities. The Mai Tai output at 790 nm is split into two beams, with 80% of the power being used to pump the OPO. The idler output at 2036 nm from OPO is doubled using a periodically poled lithium niobate (PPLN) crystal. This frequency-doubled idler beam at 1018 nm is sent through a delay line and collinearly combined with the other Mai Tai beam for CARS imaging on a laser-scanning microscope. This Mai Tai beam is also used for multiphoton fluorescence and second harmonic generation (SHG) imaging. The signal output at 1290 nm from OPO is used for SHG and third-harmonic generation (THG) imaging. External detectors are installed for both forward and backward detection, whereas two internal lamda-scan detectors are employed for microspectroscopy analysis. This new system allows vibrationally resonant CARS imaging of lipid bodies, SHG imaging of collagen fibers, and multiphoton fluorescence analysis in fresh tissues. As a preliminary application, the effect of diacylglycerol acyltransferase 1 (DGAT1) deficiency on liver lipid metabolism in mice was investigated.

  13. Nanoparticle-assisted-multiphoton microscopy for in vivo brain imaging of mice

    NASA Astrophysics Data System (ADS)

    Qian, Jun

    2015-03-01

    Neuro/brain study has attracted much attention during past few years, and many optical methods have been utilized in order to obtain accurate and complete neural information inside the brain. Relying on simultaneous absorption of two or more near-infrared photons by a fluorophore, multiphoton microscopy can achieve deep tissue penetration and efficient light detection noninvasively, which makes it very suitable for thick-tissue and in vivo bioimaging. Nanoparticles possess many unique optical and chemical properties, such as anti-photobleaching, large multiphoton absorption cross-section, and high stability in biological environment, which facilitates their applications in long-term multiphoton microscopy as contrast agents. In this paper, we will introduce several typical nanoparticles (e.g. organic dye doped polymer nanoparticles and gold nanorods) with high multiphoton fluorescence efficiency. We further applied them in two- and three-photon in vivo functional brain imaging of mice, such as brain-microglia imaging, 3D architecture reconstruction of brain blood vessel, and blood velocity measurement.

  14. Enabling Multiphoton and Second Harmonic Generation Imaging in Paraffin-Embedded and Histologically Stained Sections

    PubMed Central

    Monaghan, Michael G.; Kroll, Sebastian; Brucker, Sara Y.

    2016-01-01

    Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embedded sections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples. PMID:27018844

  15. Optical Spectroscopy and Multiphoton Imaging for the Diagnosis and Characterization of Hyperplasias in the Mouse Mammary

    DTIC Science & Technology

    2006-09-01

    was inhibited with 3 - bromopyruvate , which inhibits glyceraldehyde- 3 -phosphate dehydrogenase and 3 -phosphoglycerate kinase in a competitive manner (8...consistent with FAD fluorescence (12). Multiphoton FLIM of NADH showed that 3 - bromopyruvate caused an increase in the fluorescence lifetime of protein...images from 4 dishes), cells treated with 3 - bromopyruvate (n=6 images from 2 dishes), which inhibits glycolysis, and cells treated with CoCl2 (n=6

  16. Image segmentation for integrated multiphoton microscopy and reflectance confocal microscopy imaging of human skin in vivo

    PubMed Central

    Chen, Guannan; Lui, Harvey

    2015-01-01

    Background Non-invasive cellular imaging of the skin in vivo can be achieved in reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) modalities to yield complementary images of the skin based on different optical properties. One of the challenges of in vivo microscopy is the delineation (i.e., segmentation) of cellular and subcellular architectural features. Methods In this work we present a method for combining watershed and level-set models for segmentation of multimodality images obtained by an integrated MPM and RCM imaging system from human skin in vivo. Results Firstly, a segmentation model based on watershed is introduced for obtaining the accurate structure of cell borders from the RCM image. Secondly,, a global region based energy level-set model is constructed for extracting the nucleus of each cell from the MPM image. Thirdly, a local region-based Lagrange Continuous level-set approach is used for segmenting cytoplasm from the MPM image. Conclusions Experimental results demonstrated that cell borders from RCM image and boundaries of cytoplasm and nucleus from MPM image can be obtained by our segmentation method with better accuracy and effectiveness. We are planning to use this method to perform quantitative analysis of MPM and RCM images of in vivo human skin to study the variations of cellular parameters such as cell size, nucleus size and other mophormetric features with skin pathologies. PMID:25694949

  17. In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope

    NASA Astrophysics Data System (ADS)

    Brown, Christopher M.; Rivera, David R.; Pavlova, Ina; Ouzounov, Dimitre G.; Williams, Wendy O.; Mohanan, Sunish; Webb, Watt W.; Xu, Chris

    2012-04-01

    We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 μm by 115 μm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ~75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.

  18. In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

    NASA Astrophysics Data System (ADS)

    Lai, Zhenhua; Gu, Zetong; Abbas, Saleh; Lowe, Jared; Sierra, Heidy; Rajadhyaksha, Milind; DiMarzio, Charles

    2014-03-01

    The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

  19. Using multiphoton fluorescence lifetime imaging to characterize liver damage and fluorescein disposition in liver in vivo

    NASA Astrophysics Data System (ADS)

    Thorling, Camilla A.; Studier, Hauke; Crawford, Darrell; Roberts, Michael S.

    2016-03-01

    Liver disease is the fifth most common cause of death and unlike many other major causes of mortality, liver disease rates are increasing rather than decreasing. There is no ideal measurement of liver disease and although biopsies are the gold standard, this only allows for a spot examination and cannot follow dynamic processes of the liver. Intravital imaging has the potential to extract detailed information over a larger sampling area continuously. The aim of this project was to investigate whether multiphoton and fluorescence lifetime imaging microscopy could detect early liver damage and to assess whether it could detect changes in metabolism of fluorescein in normal and diseased livers. Four experimental groups were used in this study: 1) control; 2) ischemia reperfusion injury; 3) steatosis and 4) steatosis with ischemia reperfusion injury. Results showed that multiphoton microscopy could visualize morphological changes such as decreased fluorescence of endogenous fluorophores and the presence of lipid droplets, characteristic of steatosis. Fluorescence lifetime imaging microscopy showed increase in NADPH in steatosis with and without ischemia reperfusion injury and could detect changes in metabolism of fluorescein to fluorescein monoglurcuronide, which was impaired in steatosis with ischemia reperfusion injury. These results concluded that the combination of multiphoton microscopy and fluorescence lifetime imaging is a promising method of assessing early stage liver damage and that it can be used to study changes in drug metabolism in the liver as an indication of liver disease and has the potential to replace the traditional static liver biopsy currently used.

  20. Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores.

    PubMed

    Chen, Zhixing; Wei, Lu; Zhu, Xinxin; Min, Wei

    2012-08-13

    It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

  1. In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems

    PubMed Central

    Huland, David M.; Brown, Christopher M.; Howard, Scott S.; Ouzounov, Dimitre G.; Pavlova, Ina; Wang, Ke; Rivera, David R.; Webb, Watt W.; Xu, Chris

    2012-01-01

    We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s. The lateral and axial resolution in water is 0.85 µm and 7.4 µm respectively. In vivo images of unstained tissues in live, anesthetized rats using the portable device are presented. These results show great promise for GRIN endoscopy to be used clinically. PMID:22567597

  2. Rapid volumetric temporal focusing multiphoton microscopy of neural activity: theory, image processing, and experimental realization

    NASA Astrophysics Data System (ADS)

    Dana, Hod; Marom, Anat; Kruger, Nimrod; Ellman, Aviv; Shoham, Shy

    2012-03-01

    The development of rapid volumetric imaging systems for functional multiphoton microscopy is essential for dynamical imaging of large-scale neuronal network activity. Here, we introduce a line-illuminating temporal-focusing microscope capable of rapid three-dimensional imaging at 10-20 volumes/sec, and study the system's characteristics both theoretically and experimentally. We demonstrate that our system is capable of functional volumetric calcium imaging of distributed neuronal activity patterns, and introduce a computational strategy for activity reconstruction in strongly scattering media.

  3. Compact fixed wavelength femtosecond oscillators for multi-photon imaging

    NASA Astrophysics Data System (ADS)

    Hakulinen, T.; Klein, J.; Zadoyan, R.; Baldacchini, T.; Franke, T.

    2015-03-01

    In recent years two-photon microscopy with fixed-wavelength has raised increasing interest in life-sciences: Two-photon (2P) absorption spectra of common dyes are broader than single-photon ones. Therefore, excitation of several dyes simultaneously with a single IR laser wavelength is feasible and could be seen as an advantage in 2P microscopy. We used pulsed fixed-wavelength infrared lasers with center wavelength at 1040 nm, for two-photon microscopy in a variety of biologically relevant samples, among these a mouse brain sample, a mouse artery (within the animal, acute preparation), and a preparation of mouse bladder. The 1040 nm laser proved to be efficient not only in exciting fluorescence from yellow fluorescent protein (YFP) and red fluorescent dyes, but also for second harmonic generation (SHG) signals from muscle tissue and collagen. With this work we demonstrate that economical, small-footprint fixedwavelength lasers can present an interesting alternative to tunable lasers that are commonly used in multiphoton microscopy.

  4. Multiphoton Imaging of the Glomerular Permeability of Angiotensinogen

    PubMed Central

    Nakano, Daisuke; Kobori, Hiroyuki; Burford, James L.; Gevorgyan, Haykanush; Seidel, Saskia; Hitomi, Hirofumi; Nishiyama, Akira

    2012-01-01

    Patients and animals with renal injury exhibit increased urinary excretion of angiotensinogen. Although increased tubular synthesis of angiotensinogen contributes to the increased excretion, we do not know to what degree glomerular filtration of systemic angiotensinogen, especially through an abnormal glomerular filtration barrier, contributes to the increase in urinary levels. Here, we used multiphoton microscopy to visualize and quantify the glomerular permeability of angiotensinogen in the intact mouse and rat kidney. In healthy mice and Munich-Wistar-Frömter rats at the early stage of glomerulosclerosis, the glomerular sieving coefficient of systemically infused Atto565-labeled human angiotensinogen (Atto565-hAGT), which rodent renin cannot cleave, was only 25% of the glomerular sieving coefficient of albumin, and its urinary excretion was undetectable. In a more advanced phase of kidney disease, the glomerular permeability of Atto565-hAGT was slightly higher but still very low. Furthermore, unlike urinary albumin, the significantly higher urinary excretion of endogenous rat angiotensinogen did not correlate with either the Atto565-hAGT or Atto565-albumin glomerular sieving coefficients. These results strongly suggest that the vast majority of urinary angiotensinogen originates from the tubules rather than glomerular filtration. PMID:22997258

  5. Multiphoton imaging of the glomerular permeability of angiotensinogen.

    PubMed

    Nakano, Daisuke; Kobori, Hiroyuki; Burford, James L; Gevorgyan, Haykanush; Seidel, Saskia; Hitomi, Hirofumi; Nishiyama, Akira; Peti-Peterdi, Janos

    2012-11-01

    Patients and animals with renal injury exhibit increased urinary excretion of angiotensinogen. Although increased tubular synthesis of angiotensinogen contributes to the increased excretion, we do not know to what degree glomerular filtration of systemic angiotensinogen, especially through an abnormal glomerular filtration barrier, contributes to the increase in urinary levels. Here, we used multiphoton microscopy to visualize and quantify the glomerular permeability of angiotensinogen in the intact mouse and rat kidney. In healthy mice and Munich-Wistar-Frömter rats at the early stage of glomerulosclerosis, the glomerular sieving coefficient of systemically infused Atto565-labeled human angiotensinogen (Atto565-hAGT), which rodent renin cannot cleave, was only 25% of the glomerular sieving coefficient of albumin, and its urinary excretion was undetectable. In a more advanced phase of kidney disease, the glomerular permeability of Atto565-hAGT was slightly higher but still very low. Furthermore, unlike urinary albumin, the significantly higher urinary excretion of endogenous rat angiotensinogen did not correlate with either the Atto565-hAGT or Atto565-albumin glomerular sieving coefficients. These results strongly suggest that the vast majority of urinary angiotensinogen originates from the tubules rather than glomerular filtration.

  6. A novel multiphoton microscopy images segmentation method based on superpixel and watershed.

    PubMed

    Wu, Weilin; Lin, Jinyong; Wang, Shu; Li, Yan; Liu, Mingyu; Liu, Gaoqiang; Cai, Jianyong; Chen, Guannan; Chen, Rong

    2016-04-19

    Multiphoton microscopy (MPM) imaging technique based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker-controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness.

  7. Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates

    PubMed Central

    Botcherby, Edward J.; Smith, Christopher W.; Kohl, Michael M.; Débarre, Delphine; Booth, Martin J.; Juškaitis, Rimas; Paulsen, Ole; Wilson, Tony

    2012-01-01

    Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level. PMID:22315405

  8. Spectrally-encoded color imaging

    PubMed Central

    Kang, DongKyun; Yelin, Dvir; Bouma, Brett E.; Tearney, Guillermo J.

    2010-01-01

    Spectrally-encoded endoscopy (SEE) is a technique for ultraminiature endoscopy that encodes each spatial location on the sample with a different wavelength. One limitation of previous incarnations of SEE is that it inherently creates monochromatic images, since the spectral bandwidth is expended in the spatial encoding process. Here we present a spectrally-encoded imaging system that has color imaging capability. The new imaging system utilizes three distinct red, green, and blue spectral bands that are configured to illuminate the grating at different incident angles. By careful selection of the incident angles, the three spectral bands can be made to overlap on the sample. To demonstrate the method, a bench-top system was built, comprising a 2400-lpmm grating illuminated by three 525-μm-diameter beams with three different spectral bands. Each spectral band had a bandwidth of 75 nm, producing 189 resolvable points. A resolution target, color phantoms, and excised swine small intestine were imaged to validate the system's performance. The color SEE system showed qualitatively and quantitatively similar color imaging performance to that of a conventional digital camera. PMID:19688002

  9. Label-free imaging of rat spinal cords based on multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liao, Chenxi; Wang, Zhenyu; Zhou, Linquan; Zhu, Xiaoqin; Liu, Wenge; Chen, Jianxin

    2016-10-01

    As an integral part of the central nervous system, the spinal cord is a communication cable between the body and the brain. It mainly contains neurons, glial cells, nerve fibers and fiber tracts. The recent development of the optical imaging technique allows high-resolution imaging of biological tissues with the great potential for non-invasively looking inside the body. In this work, we evaluate the imaging capacity of multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) for the cells and extracellular matrix in the spinal cord at molecular level. Rat spinal cord tissues were sectioned and imaged by MPM to demonstrate that MPM is able to show the microstructure including white matter, gray matter, ventral horns, dorsal horns, and axons based on the distinct intrinsic sources in each region of spinal cord. In the high-resolution and high-contrast MPM images, the cell profile can be clearly identified as dark shadows caused by nuclei and encircled by cytoplasm. The nerve fibers in white matter region emitted both SHG and TPEF signals. The multiphoton microscopic imaging technique proves to be a fast and effective tool for label-free imaging spinal cord tissues, based on endogenous signals in biological tissue. It has the potential to extend this optical technique to clinical study, where the rapid and damage-free imaging is needed.

  10. ENVIRONMENTAL APPLICATIONS OF SPECTRAL IMAGING

    EPA Science Inventory

    The utility of remote sensing using spectral imaging is just being realized through the investigation to a wide variety of environmental issues. Improved spectral and spatial resolution is very important to the detection of effects once regarded as unobservable. A current researc...

  11. Rayleigh imaging in spectral mammography

    NASA Astrophysics Data System (ADS)

    Berggren, Karl; Danielsson, Mats; Fredenberg, Erik

    2016-03-01

    Spectral imaging is the acquisition of multiple images of an object at different energy spectra. In mammography, dual-energy imaging (spectral imaging with two energy levels) has been investigated for several applications, in particular material decomposition, which allows for quantitative analysis of breast composition and quantitative contrast-enhanced imaging. Material decomposition with dual-energy imaging is based on the assumption that there are two dominant photon interaction effects that determine linear attenuation: the photoelectric effect and Compton scattering. This assumption limits the number of basis materials, i.e. the number of materials that are possible to differentiate between, to two. However, Rayleigh scattering may account for more than 10% of the linear attenuation in the mammography energy range. In this work, we show that a modified version of a scanning multi-slit spectral photon-counting mammography system is able to acquire three images at different spectra and can be used for triple-energy imaging. We further show that triple-energy imaging in combination with the efficient scatter rejection of the system enables measurement of Rayleigh scattering, which adds an additional energy dependency to the linear attenuation and enables material decomposition with three basis materials. Three available basis materials have the potential to improve virtually all applications of spectral imaging.

  12. Real-time in vivo imaging collagen in lymphedematous skin using multiphoton microscopy.

    PubMed

    Wu, Xiufeng; Zhuo, Shuangmu; Chen, Jianxin; Liu, Ningfei

    2011-01-01

    Changes of dermal collagen are characteristic for chronic lymphedema. To evaluate these changes, a real-time imaging based on two-photon excited fluorescence and second-harmonic generation was developed for investigating collagen of lymphedematous mouse and rat tail skin in vivo. Our findings showed that the technique could image the morphological changes and distribution of collagen in lymphedematous mouse and rat tail skin in vivo. More importantly, it may allow visualization of dynamic collagen alteration during the progression of lymphedema. Our findings demonstrated that multiphoton microscopy may have potential in a clinical setting as an in vivo diagnostic and monitoring system for therapy in lymphology.

  13. In vivo multiphoton imaging of the cornea: polarization-resolved second harmonic generation from stromal collagen

    NASA Astrophysics Data System (ADS)

    Latour, G.; Gusachenko, I.; Kowalczuk, L.; Lamarre, I.; Schanne-Klein, M.-C.

    2012-03-01

    Multiphoton microscopy provides specific and contrasted images of unstained collagenous tissues such as tendons or corneas. Polarization-resolved second harmonic generation (SHG) measurements have been implemented in a laserscanning multiphoton microscope. Distortion of the polarimetric response due to birefringence and diattenuation during propagation of the laser excitation has been shown in rat-tail tendons. A model has been developed to account for these effects and correct polarization-resolved SHG images in thick tissues. This new modality is then used in unstained human corneas to access two quantitative parameters: the fibrils orientation within the collagen lamellae and the ratio of the main second-order nonlinear tensorial components. Orientation maps obtained from polarization resolution of the trans-detected SHG images are in good agreement with the striated features observed in the raw images. Most importantly, polarization analysis of the epi-detected SHG images also enables to map the fibrils orientation within the collagen lamellae while epi-detected SHG images of corneal stroma are spatially homogenous and do not enable direct visualization of the fibrils orientation. Depth profiles of the polarimetric SHG response are also measured and compared to models accounting for orientation changes of the collagen lamellae within the focal volume. Finally, in vivo polarization-resolved SHG is performed in rat corneas and structural organization of corneal stroma is determined using epi-detected signals.

  14. Multiphoton imaging using a Ti:sapphire regenerative amplifier

    NASA Astrophysics Data System (ADS)

    Theer, Patrick; Hasan, Mazahir T.; Denk, Winfried T.

    2003-10-01

    It is shown that two-photon fluorescence images can be obtained almost throughout the entire grey-matter layers of the mouse neocortex by using optically amplified femtosecond pulses. The maximum imaging depth is not limited by the available excitation power but instead by the generation of out-of focus fluorescence.

  15. Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging

    PubMed Central

    Stoltzfus, Caleb R.; Rebane, Aleksander

    2016-01-01

    We study the optimal conditions for high throughput two-photon excited fluorescence (2PEF) and three-photon excited fluorescence (3PEF) imaging using femtosecond lasers. We derive relations that allow maximization of the rate of imaging depending on the average power, pulse repetition rate, and noise characteristics of the laser, as well as on the size and structure of the sample. We perform our analysis using ~100 MHz, ~1 MHz and 1 kHz pulse rates and using both a tightly-focused illumination beam with diffraction-limited image resolution, as well loosely focused illumination with a relatively low image resolution, where the latter utilizes separate illumination and fluorescence detection beam paths. Our theoretical estimates agree with the experiments, which makes our approach especially useful for optimizing high throughput imaging of large samples with a field-of-view up to 10x10 cm2. PMID:27231620

  16. Fast volumetric imaging with patterned illumination via digital micro-mirror device-based temporal focusing multiphoton microscopy

    PubMed Central

    Chang, Chia-Yuan; Hu, Yvonne Yuling; Lin, Chun-Yu; Lin, Cheng-Han; Chang, Hsin-Yu; Tsai, Sheng-Feng; Lin, Tzu-Wei; Chen, Shean-Jen

    2016-01-01

    Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination. PMID:27231617

  17. Multiphoton fluorescence lifetime imaging shows spatial segregation of secondary metabolites in Eucalyptus secretory cavities.

    PubMed

    Heskes, A M; Lincoln, C N; Goodger, J Q D; Woodrow, I E; Smith, T A

    2012-07-01

    Multiphoton fluorescence lifetime imaging provides an excellent tool for imaging deep within plant tissues while providing a means to distinguish between fluorophores with high spatial and temporal resolution. Ideal candidates for the application of multiphoton fluorescence lifetime imaging to plants are the embedded secretory cavities found in numerous species because they house complex mixtures of secondary metabolites within extracellular lumina. Previous investigations of this type of structure have been restricted by the use of sectioned material resulting in the loss of lumen contents and often disorganization of the delicate secretory cells; thus it is not known if there is spatial segregation of secondary metabolites within these structures. In this paper, we apply multiphoton fluorescence lifetime imaging to investigate the spatial arrangement of metabolites within intact secretory cavities isolated from Eucalyptus polybractea R.T. Baker leaves. The secretory cavities of this species are abundant (up to 10 000 per leaf), large (up to 6 nL) and importantly house volatile essential oil rich in the monoterpene 1,8-cineole, together with an immiscible, non-volatile component comprised largely of autofluorescent oleuropeic acid glucose esters. We have been able to optically section into the lumina of secretory cavities to a depth of ∼80 μm, revealing a unique spatial organization of cavity metabolites whereby the non-volatile component forms a layer between the secretory cells lining the lumen and the essential oil. This finding could be indicative of a functional role of the non-volatile component in providing a protective region of low diffusivity between the secretory cells and potentially autotoxic essential oil.

  18. Two-color temporal focusing multiphoton excitation imaging with tunable-wavelength excitation

    NASA Astrophysics Data System (ADS)

    Lien, Chi-Hsiang; Abrigo, Gerald; Chen, Pei-Hsuan; Chien, Fan-Ching

    2017-02-01

    Wavelength tunable temporal focusing multiphoton excitation microscopy (TFMPEM) is conducted to visualize optical sectioning images of multiple fluorophore-labeled specimens through the optimal two-photon excitation (TPE) of each type of fluorophore. The tunable range of excitation wavelength was determined by the groove density of the grating, the diffraction angle, the focal length of lenses, and the shifting distance of the first lens in the beam expander. Based on a consideration of the trade-off between the tunable-wavelength range and axial resolution of temporal focusing multiphoton excitation imaging, the presented system demonstrated a tunable-wavelength range from 770 to 920 nm using a diffraction grating with groove density of 830 lines/mm. TPE fluorescence imaging examination of a fluorescent thin film indicated that the width of the axial confined excitation was 3.0±0.7 μm and the shifting distance of the temporal focal plane was less than 0.95 μm within the presented wavelength tunable range. Fast different wavelength excitation and three-dimensionally rendered imaging of Hela cell mitochondria and cytoskeletons and mouse muscle fibers were demonstrated. Significantly, the proposed system can improve the quality of two-color TFMPEM images through different excitation wavelengths to obtain higher-quality fluorescent signals in multiple-fluorophore measurements.

  19. Snapshot Raman Spectral Imager

    DTIC Science & Technology

    2010-03-31

    5 P. J. Treado, M. P. Nelson, R. Schweitzer, C. Gardner, and R. Wentworth, "Standoff Raman Hyperspectral Imaging Detection of...cope with target signatures in a wide variety of backgrounds. While the majority of Raman systems available are point-based, having imaging...for the proposed Phase I architecture. ................................................................................... 16 Figure 6: (a) Target

  20. Characterization of multiphoton photoacoustic spectroscopy for subsurface brain tissue diagnosis and imaging

    NASA Astrophysics Data System (ADS)

    Dahal, Sudhir; Cullum, Brian M.

    2016-04-01

    The development and demonstration of a multiphoton photoacoustic imaging technique capable of providing high spatial resolution chemical images of subsurface tissue components as deep as 1.4 cm below the tissue surface is described. By combining multiphoton excitation in the diagnostic window (650 to 1100 nm), with ultrasonic detection of nonradiative relaxation events, it is possible to rapidly reconstruct three-dimensional, chemical specific, images of samples underneath overlying structures as well as chemical species of the same material. Demonstration of this technique for subsurface tissue differentiation is shown, with the ability to distinguish between grade III astrocytoma tissue and adjacent healthy tissue in blind studies. By employing photoacoustic signal detection, the high nonradiative relaxation rates of most biological tissue components (>90%) and the minimal signal attenuation of the resulting ultrasound compensate for excitation efficiency losses associated with two-photon absorption. Furthermore, the two-photon absorption process results in a highly localized excitation volume (ca., 60 μm). Characterization of the probing depth, spatial resolution, and ability to image through overlying structures is also demonstrated in this paper using tissue phantoms with well-characterized optical scattering properties, mimicking those of tissues.

  1. Multimodal, multiphoton microscopy and image correlation analysis for characterizing corneal thermal damage

    NASA Astrophysics Data System (ADS)

    Lo, Wen; Chang, Yu-Lin; Liu, Jia-Shiu; Hseuh, Chiu-Mei; Hovhannisyan, Vladimir; Chen, Shean-Jen; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2009-09-01

    We used the combination of multiphoton autofluorescence (MAF), forward second-harmonic generation (FWSHG), and backward second-harmonic generation (BWSHG) imaging for the qualitative and quantitative characterization of thermal damage of ex vivo bovine cornea. We attempt to characterize the structural alterations by qualitative MAF, FWSHG, and BWSHG imaging in the temperature range of 37 to 90°C. In addition to measuring the absolute changes in the three types of signals at the stromal surface, we also performed image correlation analysis between FWSHG and BWSHG and demonstrate that with increasing thermal damage, image correlation between FWSHG and BWSHG significantly increases. Our results show that while MAF and BWSHG intensities may be used as preliminary indicators of the extent of corneal thermal damage, the most sensitive measures are provided by the decay in FWSHG intensity and the convergence of FWSHG and BWSHG images.

  2. Quantitative characterization of articular cartilage using Mueller matrix imaging and multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Ellingsen, Pa˚L. Gunnar; Lilledahl, Magnus Borstad; Aas, Lars Martin Sandvik; Davies, Catharina De Lange; Kildemo, Morten

    2011-11-01

    The collagen meshwork in articular cartilage of chicken knee is characterized using Mueller matrix imaging and multiphoton microscopy. Direction and degree of dispersion of the collagen fibers in the superficial layer are found using a Fourier transform image-analysis technique of the second-harmonic generated image. Mueller matrix images are used to acquire structural data from the intermediate layer of articular cartilage where the collagen fibers are too small to be resolved by optical microscopy, providing a powerful multimodal measurement technique. Furthermore, we show that Mueller matrix imaging provides more information about the tissue compared to standard polarization microscopy. The combination of these techniques can find use in improved diagnosis of diseases in articular cartilage, improved histopathology, and additional information for accurate biomechanical modeling of cartilage.

  3. Simultaneous measurement of neural spike recordings and multi-photon calcium imaging in neuroblastoma cells.

    PubMed

    Kim, Suhwan; Jung, Unsang; Baek, Juyeong; Kang, Shinwon; Kim, Jeehyun

    2012-11-08

    This paper proposes the design and implementation of a micro-electrode array (MEA) for neuroblastoma cell culturing. It also explains the implementation of a multi-photon microscope (MPM) customized for neuroblastoma cell excitation and imaging under ambient light. Electrical signal and fluorescence images were simultaneously acquired from the neuroblastoma cells on the MEA. MPM calcium images of the cultured neuroblastoma cell on the MEA are presented and also the neural activity was acquired through the MEA recording. A calcium green-1 (CG-1) dextran conjugate of 10,000 D molecular weight was used in this experiment for calcium imaging. This study also evaluated the calcium oscillations and neural spike recording of neuroblastoma cells in an epileptic condition. Based on our observation of neural spikes in neuroblastoma cells with our proposed imaging modality, we report that neuroblastoma cells can be an important model for epileptic activity studies.

  4. Multiphoton fluorescence microscopic imaging through double-layer turbid tissue media

    NASA Astrophysics Data System (ADS)

    Deng, Xiaoyuan; Gan, Xiaosong; Gu, Min

    2002-04-01

    Image formation in multiphoton fluorescence microscopy through double-layer turbid tissue media is investigated using Monte Carlo simulation. With the help of the concept of the effective point spread function, the relationship of image resolution and signal level to the thickness and scattering properties of the double-layer turbid media under single-, two-, and three-photon excitation is revealed. Results show that for a double-layer turbid medium of a given thickness, small particles in the top layer result in a quicker degradation of signal level than large particles in the top layer. This model is then applied to study the penetration depth of multiphoton fluorescence microscopy through human skin tissue which exhibits a layered structure. It is predicated that using 3p excitation leads to a signal level up to two orders of magnitude higher than that under 2p excitation, while diffraction-limited image resolution can be maintained for skin tissue of thickness up to 500 μm.

  5. Chronic imaging of amyloid plaques in the live mouse brain using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Bacskai, Brian J.; Kajdasz, Stephen T.; Christie, R. H.; Zipfel, Warren R.; Williams, Rebecca M.; Kasischke, Karl A.; Webb, Watt W.; Hyman, B. T.

    2001-04-01

    Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.

  6. Imaging sulfur mustard lesions in human epidermal tissues and keratinocytes by confocal and multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert; Madren-Whalley, Janna S.

    2002-06-01

    Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) 6 integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) 6 integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.

  7. Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy.

    PubMed

    Shelton, Ryan L; Mattison, Scott P; Applegate, Brian E

    2014-10-01

    Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high-resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes.

  8. 5-HT spatial distribution imaging with multiphoton excitation of 5-HT correlative visible fluorescence in live cells

    NASA Astrophysics Data System (ADS)

    Zhang, Zhihong; Zeng, Shaoqun; Liu, Yafeng; Zhou, Wei; Chen, Tongsheng; Luo, Qingming

    2002-04-01

    The autofluorescence of 5-Hydroxytryptamine (5-HT) loaded rat mucosal mast cells (RBL-2H3 cells) is imaged with multiphoton excitation laser scanning microscope (MPELSM). 5-HT correlative visible fluorescence (Fco-vis) excited with 740-nm multiphoton excitation is observed in live cells for the first time, and the generating mechanism of 5-HT Fco-vis is studied. The spatial distribution of 5-HT in live cells is imaged at high spatial resolution in our experiment, which provides a new way to study the correlation between 5-HT spatial distribution and content, and the cellular functional state in live tissue or cells.

  9. Hybrid Multiphoton Volumetric Functional Imaging of Large Scale Bioengineered Neuronal Networks

    PubMed Central

    Paluch, Shir; Dvorkin, Roman; Brosh, Inbar; Shoham, Shy

    2014-01-01

    Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bio-engineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes/sec of structures with mm-scale dimensions containing a network of over 1000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances. PMID:24898000

  10. Multiphoton microscopic imaging of fibrotic focus in invasive ductal carcinoma of the breast

    NASA Astrophysics Data System (ADS)

    Chen, Sijia; Nie, Yuting; Lian, Yuane; Wu, Yan; Fu, Fangmeng; Wang, Chuan; Zhuo, Shuangmu; Chen, Jianxin

    2014-11-01

    During the proliferation of breast cancer, the desmoplastic can evoke a fibrosis response by invading healthy tissue. Fibrotic focus (FF) in invasive ductal carcinoma (IDC) of the breast had been reported to be associated with significantly poorer survival rate than IDC without FF. As an important prognosis indicator, it's difficult to obtain the exact fibrotic information from traditional detection method such as mammography. Multiphoton imaging based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) has been recently employed for microscopic examination of unstained tissue. In this study, multiphoton microscopy (MPM) was used to image the fibrotic focus in invasive ductal carcinoma tissue. The morphology and distribution of collagen in fibrotic focus can be demonstrated by the SHG signal. Variation of collagen between IDC with and without FF will be examined and further characterized, which may be greatly related to the metastasis of breast cancer. Our result suggested that the MPM can be efficient in identifying and locating the fibrotic focus in IDC. Combining with the pathology analysis and other detecting methods, MPM owns potential in becoming an advanced histological tool for detecting the fibrotic focus in IDC and collecting prognosis information, which may guide the subsequent surgery option and therapy procedure for patients.

  11. Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks

    NASA Astrophysics Data System (ADS)

    Dana, Hod; Marom, Anat; Paluch, Shir; Dvorkin, Roman; Brosh, Inbar; Shoham, Shy

    2014-06-01

    Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bioengineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes per second of structures with mm-scale dimensions containing a network of over 1,000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances.

  12. Photoelectron imaging following 2 + 1 multiphoton excitation of HBr.

    PubMed

    Romanescu, Constantin; Loock, Hans-Peter

    2006-07-07

    The photodissociation and photoionization dynamics of HBr via low-n Rydberg and ion-pair states was studied by using 2 + 1 REMPI spectroscopy and velocity map imaging of photoelectrons. Two-photon excitation at about 9.4-10 eV was used to prepare rotationally selected excited states. Following absorption of the third photon the unperturbed F (1)Delta(2) and i (3)Delta(2) states ionize directly into the ground vibrational state of the molecular ion according to the Franck-Condon principle and upon preservation of the ion core. In case of the V (1)Sigma(+)(0(+)) ion-pair state and the perturbed E (1)Sigma(+)(0(+)), g (3)Sigma(-)(0(+)), and H (1)Sigma(+)(0(+)) Rydberg states the absorption of the third photon additionally results in a long vibrational progression of HBr(+) in the X (2)Pi state as well as formation of electronically excited atomic photofragments. The vibrational excitation of the molecular ion is explained by autoionization of repulsive superexcited states into the ground state of the molecular ion. In contrast to HCl, the perturbed Rydberg states of HBr show strong participation of the direct ionization process, with ionic core preservation.

  13. Superresolving Imaging of Arbitrary One-Dimensional Arrays of Thermal Light Sources Using Multiphoton Interference

    NASA Astrophysics Data System (ADS)

    Classen, Anton; Waldmann, Felix; Giebel, Sebastian; Schneider, Raimund; Bhatti, Daniel; Mehringer, Thomas; von Zanthier, Joachim

    2016-12-01

    We propose to use multiphoton interferences of photons emitted from statistically independent thermal light sources in combination with linear optical detection techniques to reconstruct, i.e., image, arbitrary source geometries in one dimension with subclassical resolution. The scheme is an extension of earlier work [S. Oppel et al., Phys. Rev. Lett. 109, 233603 (2012)], where N regularly spaced sources in one dimension were imaged by use of the N th-order intensity correlation function. Here, we generalize the scheme to reconstruct any number of independent thermal light sources at arbitrary separations in one dimension, exploiting intensity correlation functions of order m ≥3 . We present experimental results confirming the imaging protocol and provide a rigorous mathematical proof for the obtained subclassical resolution.

  14. Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Yu, Jiun-Yann; Kuo, Chun-Hung; Holland, Daniel B.; Chen, Yenyu; Ouyang, Mingxing; Blake, Geoffrey A.; Zadoyan, Ruben; Guo, Chin-Lin

    2011-11-01

    Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.

  15. Label-free in vivo imaging of Drosophila melanogaster by multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Chiao-Ying; Hovhannisyan, Vladimir; Wu, June-Tai; Lin, Sung-Jan; Lin, Chii-Wann; Chen, Jyh-Horng; Dong, Chen-Yuan

    2008-02-01

    The fruit fly Drosophila melanogaster is one of the most valuable organisms in genetic and developmental biology studies. Drosophila is a small organism with a short life cycle, and is inexpensive and easy to maintain. The entire genome of Drosophila has recently been sequenced (cite the reference). These advantages make fruit fly an attractive model organism for biomedical researches. Unlike humans, Drosophila can be subjected to genetic manipulation with relative ease. Originally, Drosophila was mostly used in classical genetics studies. In the model era of molecular biology, the fruit fly has become a model organ for developmental biology researches. In the past, numerous molecularly modified mutants with well defined genetic defects affecting different aspects of the developmental processes have been identified and studied. However, traditionally, the developmental defects of the mutant flies are mostly examined in isolated fixed tissues which preclude the observation of the dynamic interaction of the different cell types and the extracellular matrix. Therefore, the ability to image different organelles of the fruit fly without extrinsic labeling is invaluable for Drosophila biology. In this work, we successfully acquire in vivo images of both developing muscles and axons of motor neurons in the three larval stages by using the minimially invasive imaging modality of multiphoton (SHG) microscopy. We found that while SHG imaging is useful in revealing the muscular architecture of the developing larva, it is the autofluorescence signal that allows label-free imaging of various organelles to be achieved. Our results demonstrate that multiphoton imaging is a powerful technique for investigation the development of Drosophila.

  16. In vivo multiphoton imaging of human skin: assessment of topical corticosteroid-induced epidermis atrophy and depigmentation

    NASA Astrophysics Data System (ADS)

    Ait El Madani, Hassan; Tancrède-Bohin, Emmanuelle; Bensussan, Armand; Colonna, Anne; Dupuy, Alain; Bagot, Martine; Pena, Ana-Maria

    2012-02-01

    Multiphoton microscopy has emerged in the past decade as a promising tool for noninvasive skin imaging. Our aim was to evaluate the potential of multiphoton microscopy to detect topical corticosteroids side effects within the epidermis and to provide new insights into their dynamics. Healthy volunteers were topically treated with clobetasol propionate on a small region of their forearms under overnight occlusion for three weeks. The treated region of each patient was investigated at D0, D7, D15, D22 (end of the treatment), and D60. Our study shows that multiphoton microscopy allows for the detection of corticoid-induced epidermis modifications: thinning of stratum corneum compactum and epidermis, decrease of keratinocytes size, and changes in their morphology from D7 to D22. We also show that multiphoton microscopy enables in vivo three-dimensional (3-D) quantitative assessment of melanin content. We observe that melanin density decreases during treatment and almost completely disappears at D22. Moreover, these alterations are reversible as they are no longer present at D60. Our study demonstrates that multiphoton microscopy is a convenient and powerful tool for noninvasive 3-D dynamical studies of skin integrity and pigmentation.

  17. Second- and third-harmonic generation and multiphoton excitation fluorescence microscopy for simultaneous imaging of cardiomyocytes

    NASA Astrophysics Data System (ADS)

    Barzda, Virginijus; Greenhalgh, Catherine; Aus der Au, Juerg; Squier, Jeffrey A.; Elmore, Steven; van Beek, Johannes H.

    2004-06-01

    Simultaneous detection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence with ultrafast laser pulses from a Nd:Glass laser was used to image isolated adult rat cardiomyocytes. The simultaneous detection enabled visualization of different organelles of cardiomyocytes, based on the different contrast mechanisms. It was found that SHG signal depicted characteristic patterns of sarcomeres in a myofilament lattice. The regular pattern of the THG signal, which was anticorrelated with the SHG signal, suggested that the third harmonic is generated within mitochondria. By labeling the cardiomyocytes with the mitochondrial dye tetramethylrhodamine methyl ester (TMRM), comparisons could be made between the TMRM fluorescence, THG, and SHG images. The TMRM fluorescence had significant correlation with THG signal confirming that part of the THG signal originates from mitochondria.

  18. Quantification of scar margin in keloid different from atrophic scar by multiphoton microscopic imaging.

    PubMed

    Zhu, Xiaoqin; Zhuo, Shuangmu; Zheng, Liqin; Jiang, Xingshan; Chen, Jianxin; Lin, Bifang

    2011-01-01

    Multiphoton microscopy (MPM) was applied to examine the marginal region at dermis of keloid compared with atrophic scar. High-resolution large-area image showed an obvious boundary at the scar margin and different morphological patterns of elastin and collagen on the two sides, further visualized by the focused three-dimensional images. Content alteration of elastin or collagen between the two sides of boundary was quantified to show significant difference between keloid and atrophic scar. Owing to the raised property of keloid with overproduced collagen on the scar side, the content alteration was positive for elastin and negative for collagen. On the contrary, the content alteration was negative for elastin and positive for collagen in the atrophic scar case due to the atrophic collagen on the scar side. It indicated that examination of the scar margin by MPM may lead a new way to discriminate different types of scars and better understand the scarring mechanisms.

  19. New developments in multimodal clinical multiphoton tomography

    NASA Astrophysics Data System (ADS)

    König, Karsten

    2011-03-01

    80 years ago, the PhD student Maria Goeppert predicted in her thesis in Goettingen, Germany, two-photon effects. It took 30 years to prove her theory, and another three decades to realize the first two-photon microscope. With the beginning of this millennium, first clinical multiphoton tomographs started operation in research institutions, hospitals, and in the cosmetic industry. The multiphoton tomograph MPTflexTM with its miniaturized flexible scan head became the Prism-Award 2010 winner in the category Life Sciences. Multiphoton tomographs with its superior submicron spatial resolution can be upgraded to 5D imaging tools by adding spectral time-correlated single photon counting units. Furthermore, multimodal hybrid tomographs provide chemical fingerprinting and fast wide-field imaging. The world's first clinical CARS studies have been performed with a hybrid multimodal multiphoton tomograph in spring 2010. In particular, nonfluorescent lipids and water as well as mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen have been imaged in patients with dermatological disorders. Further multimodal approaches include the combination of multiphoton tomographs with low-resolution imaging tools such as ultrasound, optoacoustic, OCT, and dermoscopy systems. Multiphoton tomographs are currently employed in Australia, Japan, the US, and in several European countries for early diagnosis of skin cancer (malignant melanoma), optimization of treatment strategies (wound healing, dermatitis), and cosmetic research including long-term biosafety tests of ZnO sunscreen nanoparticles and the measurement of the stimulated biosynthesis of collagen by anti-ageing products.

  20. Label-free multi-photon imaging of dysplasia in Barrett’s esophagus

    PubMed Central

    Mehravar, Soroush; Banerjee, Bhaskar; Chatrath, Hemant; Amirsolaimani, Babak; Patel, Krunal; Patel, Charmi; Norwood, Robert A; Peyghambarian, Nasser; Kieu, Khanh

    2015-01-01

    Barrett’s esophagus (BE) is a metaplastic disorder where dysplastic and early cancerous changes are invisible to the naked eye and where the practice of blind biopsy is hampered by large sampling errors. Multi-photon microscopy (MPM) has emerged as an alternative solution for fast and label-free diagnostic capability for identifying the histological features with sub-micron accuracy. We developed a compact, inexpensive MPM system by using a handheld mode-locked fiber laser operating at 1560nm to study mucosal biopsies of BE. The combination of back-scattered THG, back-reflected forward THG and SHG signals generate images of cell nuclei and collagen, leading to label-free diagnosis in Barrett’s. PMID:26819824

  1. The first decade of using multiphoton microscopy for high-power kidney imaging.

    PubMed

    Peti-Peterdi, János; Burford, James L; Hackl, Matthias J

    2012-01-15

    In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans.

  2. The first decade of using multiphoton microscopy for high-power kidney imaging

    PubMed Central

    Burford, James L.; Hackl, Matthias J.

    2012-01-01

    In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans. PMID:22031850

  3. Retinal cell imaging in myopic chickens using adaptive optics multiphoton microscopy

    PubMed Central

    Bueno, Juan M.; Palacios, Raquel; Giakoumaki, Anastasia; Gualda, Emilio J.; Schaeffel, Frank; Artal, Pablo

    2014-01-01

    Abnormal eye growth induced by visual deprivation can modify the structure and density of the retinal cells. We have used an adaptive optics multiphoton microscope to image photoreceptors (PRs) and ganglion cells (GCs) at different retinal locations in unstained retinas of chicken eyes with about 10D of myopia and their normal-sighted fellow eyes. In all samples, the local averaged inter-PR distance increased with eccentricity. No significant differences in PR density were found between control and myopic eyes. GC density declined in myopic eyes compared to control eyes and the inter-cell distance increased. In normal eyes, the size of the GC cell bodies increased approximately two-fold between the area centralis and the peripheral retina. In myopic eyes, this trend was preserved but the GC bodies were larger at each retinal location, compared to control eyes. Obviously, GC morphology is changing when the retinal area is enlarged in myopic eyes. PMID:24688804

  4. Active spectral imaging and mapping

    NASA Astrophysics Data System (ADS)

    Steinvall, Ove

    2014-04-01

    Active imaging and mapping using lasers as illumination sources have been of increasing interest during the last decades. Applications range from defense and security, remote sensing, medicine, robotics, and others. So far, these laser systems have mostly been based on a fix wavelength laser. Recent advances in lasers enable emission of tunable, multiline, or broadband emission, which together with the development of array detectors will extend the capabilities of active imaging and mapping. This paper will review some of the recent work on active imaging mainly for defense and security and remote sensing applications. A short survey of basic lidar relations and present fix wavelength laser systems is followed by a review of the benefits of adding the spectral dimension to active and/or passive electro-optical systems.

  5. Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

    PubMed Central

    2011-01-01

    Background Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes. Results Here we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications. Conclusions Similar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM. PMID:21366923

  6. Flexible polygon-mirror based laser scanning microscope platform for multiphoton in-vivo imaging.

    PubMed

    Li, Y X; Gautam, V; Brüstle, A; Cockburn, I A; Daria, V R; Gillespie, C; Gaus, K; Alt, C; Lee, W M

    2017-02-06

    Commercial microscopy systems make use of tandem scanning i.e. either slow or fast scanning. We constructed, for the first time, an advanced control system capable of delivering a dynamic line scanning speed ranging from 2.7 kHz to 27 kHz and achieve variable frame rates from 5 Hz to 50 Hz (512 × 512). The dynamic scanning ability is digitally controlled by a new customized open-source software named PScan1.0. This permits manipulation of scanning rates either to gain higher fluorescence signal at slow frame rate without increasing laser power or increase frame rates to capture high speed events. By adjusting imaging speed from 40 Hz to 160 Hz, we capture a range of calcium waves and transient peaks from soma and dendrite of single fluorescence neuron (CAL-520AM). Motion artifacts arising from respiratory and cardiac motion in small animal imaging reduce quality of real-time images of single cells in-vivo. An image registration algorithm, integrated with PScan1.0, was shown to perform both real time and post-processed motion correction. The improvement is verified by quantification of blood flow rates. This work describes all the steps necessary to develop a high performance and flexible polygon-mirror based multiphoton microscope system for in-vivo biological imaging.

  7. Spectrally Adaptable Compressive Sensing Imaging System

    DTIC Science & Technology

    2014-05-01

    2D coded projections. The underlying spectral 3D data cube is then recovered using compressed sensing (CS) reconstruction algorithms which assume...introduced in [?], is a remarkable imaging architecture that allows capturing spectral imaging information of a 3D cube with just a single 2D mea...allows capturing spectral imaging information of a 3D cube with just a single 2D measurement of the coded and spectrally dispersed source field

  8. Multiphoton microscopy based cryo-imaging of inflated frozen human lung sections at -60°C in healthy and COPD lungs

    NASA Astrophysics Data System (ADS)

    Abraham, Thomas; Kayra, Damian; Zhang, Angela; Suzuki, Masaru; McDonough, John; Elliott, W. M.; Cooper, Joel D.; Hogg, James C.

    2013-02-01

    Lung is a complex gas exchanger with interfacial area (where the gas exchange takes place) is about the size of a tennis court. Respiratory function is linked to the biomechanical stability of the gas exchange or alveolar regions which directly depends on the spatial distributions of the extracellular matrix fibers such fibrillar collagens and elastin fibers. It is very important to visualize and quantify these fibers at their native and inflated conditions to have correct morphometric information on differences between control and diseased states. This can be only achieved in the ex vivo states by imaging directly frozen lung specimens inflated to total lung capacity. Multiphoton microscopy, which uses ultra-short infrared laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from endogenously fluorescent proteins (e.g. elastin) and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens in fresh human lung tissues [J. Struct. Biol. (2010)171,189-196]. Here we report for the first time 3D image data obtained directly from thick frozen inflated lung specimens (~0.7- 1.0 millimeter thick) visualized at -60°C without prior fixation or staining in healthy and diseased states. Lung specimens donated for transplantation and released for research when no appropriate recipient was identified served as controls, and diseased lung specimens donated for research by patients receiving lung transplantation for very severe COPD (n=4) were prepared as previously described [N. Engl. J. Med. (2011) 201, 1567]. Lung slices evenly spaced between apex and base were examined using multiphoton microscopy while maintained at -60°C using a temperature controlled cold stage with a temperature resolution of 0.1°C. Infrared femto-second laser pulses tuned to 880nm, dry microscopic objectives, and non-de-scanned detectors/spectrophotometer located in the reflection geometry were

  9. Minimally invasive imaging method based on second harmonic generation and multiphoton excitation fluorescence in translational respiratory research.

    PubMed

    Abraham, Thomas; Wadsworth, Samuel; Carthy, Jon M; Pechkovsky, Dmitri V; McManus, Bruce

    2011-01-01

    For translational respiratory research including in the development of clinical diagnostic tools, a minimally invasive imaging method, which can provide both cellular and extracellular structural details with sufficient specificity, sensitivity and spatial resolution, is particularly useful. Multiphoton microscopy causes excitation of endogenously fluorescent macromolecular systems and induces highly specific second harmonic generation signals from non-centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of extracellular matrix as well as the cellular morphologies in their native states. Besides briefly discussing the fundamentals of multiphoton excitation fluorescence and harmonic generation signals and the instrumentation details, this review focuses on the specific applications of these imaging modalities in lung structural imaging, particularly morphological features of alveolar structures, visualizing and quantifying extracellular matrix remodelling accompanying emphysematous destructions as well as the IPF, detecting lung cancers and the potential use in the tissue engineering applications.

  10. Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains

    NASA Astrophysics Data System (ADS)

    Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

    2014-09-01

    Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear-cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

  11. Multi-photon Imaging of Actin Filament Formation and Mitochondrial Energetics of Human ACBT Gliomas

    PubMed Central

    Hwang, Yu-Jer; Kolettis, Nomiki; Yang, Miso; Gillard, Elizabeth R.; Sanchez, Edgar; Sun, Chung-ho; Tromberg, Bruce J.; Krasieva, Tatiana B.; Lyubovitsky, Julia G.

    2011-01-01

    We studied the three-dimensional (3D) distribution of actin filaments and mitochondria in relation to ACBT glioblastoma cells migration. We embedded the cells in the spheroid form within collagen hydrogels and imaged them by in-situ multi-photon microscopy (MPM). The static 3D overlay of the distribution of actin filaments and mitochondria provided a greater understanding of cell-to-cell and cell-to-substrate interactions and morphology. While imaging mitochondria to obtain ratiometric redox index based on cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) we observed differential sensitivity of the migrating ACBT glioblastoma cells to femtosecond laser irradiation employed in MPM. We imaged actin-GFP fluorescence in live ACBT glioma cells and for the first time observed dynamic modulation of the pools of actin during migration in 3D. The MPM imaging, which probes cells directly within the 3D cancer models, could potentially aid in working out a link between the functional performance of mitochondria, actin distribution and cancer invasiveness. PMID:21143483

  12. Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains.

    PubMed

    Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

    2014-09-01

    Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear–cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

  13. [Spectral calibration of hyperspectral imager based on spectral absorption target].

    PubMed

    Gou, Zhi-Yang; Yan, Lei; Chen, Wei; Zhao, Hong-Ying; Yin, Zhong-Yi; Duan, Yi-Ni

    2013-02-01

    Retrieval of center wavelength and bandwidth is a key step for quantitative analysis of hyperspectral data. The present paper proposes a spectral calibration method of hyperspectral imager, whose spectrum covers visible and near-infrared band, using spectral absorption target. Ground calibration experiment was designed for a hyperspectral imager with a bandwidth of 6 nm. Hyperspectral imager and ASD spectrometer measured the same spectral absorption target synchronously. Reflectance spectrum was derived from the different data set. Center wavelength and bandwidth were retrieved by matching the reflectance data from hyperspectral imager and ASD spectrometer. The experiment result shows that this method can be applied in spectral calibration of hyperspectral imagers to improve the quantitative studies on hyperspectral imagery.

  14. Use of multiphoton tomography and fluorescence lifetime imaging to investigate skin pigmentation in vivo

    NASA Astrophysics Data System (ADS)

    Dancik, Yuri; Favre, Amandine; Loy, Chong Jin; Zvyagin, Andrei V.; Roberts, Michael S.

    2013-02-01

    There is a growing body of literature showing the usefulness of multiphoton tomography (MPT) and fluorescence lifetime imaging for in situ characterization of skin constituents and the ensuing development of noninvasive diagnostic tools against skin diseases. Melanin and pigmentation-associated skin cancers constitute some of the major applications. We show that MPT and fluorescence lifetime imaging can be used to measure changes in cutaneous melanin concentration and that these can be related to the visible skin color. Melanin in the skin of African, Indian, Caucasian, and Asian volunteers is detected on the basis of its emission wavelength and fluorescence lifetimes in solution and in a melanocyte-keratinocyte cell culture. Fluorescence intensity is used to characterize the melanin content and distribution as a function of skin type and depth into the skin (stratum granulosum and stratum basale). The measured fluorescence intensities in given skin types agree with melanin amounts reported by others using biopsies. Our results suggest that spatial distribution of melanin in skin can be studied using MPT and fluorescence lifetime imaging, but further studies are needed to ascertain that the method can resolve melanin amount in smaller depth intervals.

  15. Combined multiphoton imaging and automated functional enucleation of porcine oocytes using femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Kuetemeyer, Kai; Lucas-Hahn, Andrea; Petersen, Bjoern; Lemme, Erika; Hassel, Petra; Niemann, Heiner; Heisterkamp, Alexander

    2010-07-01

    Since the birth of ``Dolly'' as the first mammal cloned from a differentiated cell, somatic cell cloning has been successful in several mammalian species, albeit at low success rates. The highly invasive mechanical enucleation step of a cloning protocol requires sophisticated, expensive equipment and considerable micromanipulation skill. We present a novel noninvasive method for combined oocyte imaging and automated functional enucleation using femtosecond (fs) laser pulses. After three-dimensional imaging of Hoechst-labeled porcine oocytes by multiphoton microscopy, our self-developed software automatically identified the metaphase plate. Subsequent irradiation of the metaphase chromosomes with the very same laser at higher pulse energies in the low-density-plasma regime was used for metaphase plate ablation (functional enucleation). We show that fs laser-based functional enucleation of porcine oocytes completely inhibited the parthenogenetic development without affecting the oocyte morphology. In contrast, nonirradiated oocytes were able to develop parthenogenetically to the blastocyst stage without significant differences to controls. Our results indicate that fs laser systems have great potential for oocyte imaging and functional enucleation and may improve the efficiency of somatic cell cloning.

  16. Nanostructures based on quantum dots for application in promising methods of single- and multiphoton imaging and diagnostics

    NASA Astrophysics Data System (ADS)

    Nabiev, I. R.

    2017-01-01

    Molecules recognizing biomarkers of diseases (monoclonal antibodies (monoABs)) are often too large for biomedical applications, and the conditions that are used to bind them with nanolabels lead to disordered orientation of monoABs with respect to the nanoparticle surface. Extremely small nanoprobes, designed via oriented conjugation of quantum dots (QDs) with single-domain antibodies (sdABs) derived from the immunoglobulin of llama and produced in the E. coli culture, have a hydrodynamic diameter less than 12 nm and contain equally oriented sdAB molecules on the surface of each QD. These nanoprobes exhibit excellent specificity and sensitivity in quantitative determination of a small number of cells expressing biomarkers. In addition, the higher diffusion coefficient of sdABs makes it possible to perform immunohistochemical analysis in bulk tissue, inaccessible for conventional monoABs. The necessary conditions for implementing high-quality immunofluorescence diagnostics are a high specificity of labeling and clear differences between the fluorescence of nanoprobes and the autofluorescence of tissues. Multiphoton micros-copy with excitation in the near-IR spectral range, which is remote from the range of tissue autofluorescence excitation, makes it possible to solve this problem and image deep layers in biological tissues. The two-photon absorption cross sections of CdSe/ZnS QDs conjugated with sdABs exceed the corresponding values for organic fluorophores by several orders of magnitude. These nanoprobes provide clear discrimination between the regions of tumor and normal tissues with a ratio of the sdAB fluorescence to the tissue autofluorescence upon two-photon excitation exceeding that in the case of single-photon excitation by a factor of more than 40. The data obtained indicate that the sdAB-QD conjugates used as labels provide the same, or even better, quality as the "gold standard" of immunohistochemical diagnostics. The developed nanoprobes are expected to

  17. Video-rate resonant scanning multiphoton microscopy: An emerging technique for intravital imaging of the tumor microenvironment.

    PubMed

    Kirkpatrick, Nathaniel D; Chung, Euiheon; Cook, Daniel C; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L; Padera, Timothy P; Fukumura, Dai; Jain, Rakesh K

    2012-01-01

    The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates-only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.

  18. Staring 2-D hadamard transform spectral imager

    DOEpatents

    Gentry, Stephen M.; Wehlburg, Christine M.; Wehlburg, Joseph C.; Smith, Mark W.; Smith, Jody L.

    2006-02-07

    A staring imaging system inputs a 2D spatial image containing multi-frequency spectral information. This image is encoded in one dimension of the image with a cyclic Hadamarid S-matrix. The resulting image is detecting with a spatial 2D detector; and a computer applies a Hadamard transform to recover the encoded image.

  19. Quantum dot capped magnetite nanorings as high performance nanoprobe for multiphoton fluorescence and magnetic resonance imaging.

    PubMed

    Fan, Hai-Ming; Olivo, Malini; Shuter, Borys; Yi, Jia-Bao; Bhuvaneswari, Ramaswamy; Tan, Hui-Ru; Xing, Gui-Chuan; Ng, Cheng-Teng; Liu, Lei; Lucky, Sasidharan S; Bay, Boon-Huat; Ding, Jun

    2010-10-27

    In the present study, quantum dot (QD) capped magnetite nanorings (NRs) with a high luminescence and magnetic vortex core have been successfully developed as a new class of magnetic-fluorescent nanoprobe. Through electrostatic interaction, cationic polyethylenimine (PEI) capped QD have been firmly graft into negatively charged magnetite NRs modified with citric acid on the surface. The obtained biocompatible multicolor QD capped magnetite NRs exhibit a much stronger magnetic resonance (MR) T2* effect where the r2* relaxivity and r2*/r1 ratio are 4 times and 110 times respectively larger than those of a commercial superparamagnetic iron oxide. The multiphoton fluorescence imaging and cell uptake of QD capped magnetite NRs are also demonstrated using MGH bladder cancer cells. In particular, these QD capped magnetite NRs can escape from endosomes and be released into the cytoplasm. The obtained results from these exploratory experiments suggest that the cell-penetrating QD capped magnetite NRs could be an excellent dual-modality nanoprobe for intracellular imaging and therapeutic applications. This work has shown great potential of the magnetic vortex core based multifunctional nanoparticle as a high performance nanoprobe for biomedical applications.

  20. Imaging Mitochondrial Organization in Living Primate Oocytes and Embryos using Multiphoton Microscopy

    NASA Astrophysics Data System (ADS)

    Squirrell, J. M.; Schramm, R. D.; Paprocki, A. M.; Wokosin, D. L.; Bavister, B. D.

    2003-06-01

    We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage—in the same embryo—thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

  1. Multiphoton ionization/dissociation dynamics of formyl fluoride by velocity mapping ion imaging.

    PubMed

    Wang, Fengyan; Zhang, Yongwei; Wang, Hua; Liu, Jie; Jiang, Bo; Wang, Xiuyan; Yang, Xueming

    2009-10-21

    The dissociation dynamics of HFCO(+) ion has been studied using the velocity map ion imaging technique. The HFCO(+) ion is prepared by one-photon resonant three-photon ionization in the region of 43100-43860 cm(-1) excitation energy. The HFCO(+) ions, produced by multiphoton ionization, have sufficient internal energy to dissociate into the F and HCO(+) fragments without further absorption of another photon. Images of HCO(+) have been recorded at various excitation energies. It is noticed that the angular distributions of HCO(+) change dramatically from parallel distribution to perpendicular distribution and then back to parallel distribution in a very narrow excitation energy region of 43 473-43 500 cm(-1). Analysis of anisotropy parameters of beta(n) (n = 2, 4 and 6) reveals that the electronic states in the three-photon excitation of HFCO are mainly: HFCO(X(1)A') --> HFCO(A(1)A'') --> HFCO(A') --> HFCO(+)(A(2)A'';B(2)A'). The purely perpendicular resonant transitions are likely responsible for the perpendicular angular distribution of the HCO(+) ion fragment.

  2. Advanced multiphoton methods for in vitro and in vivo functional imaging of mouse retinal neurons (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Cohen, Noam; Schejter, Adi; Farah, Nairouz; Shoham, Shy

    2016-03-01

    Studying the responses of retinal ganglion cell (RGC) populations has major significance in vision research. Multiphoton imaging of optogenetic probes has recently become the leading approach for visualizing neural populations and has specific advantages for imaging retinal activity during visual stimulation, because it leads to reduced direct photoreceptor excitation. However, multiphoton retinal activity imaging is not straightforward: point-by-point scanning leads to repeated neural excitation while optical access through the rodent eye in vivo has proven highly challenging. Here, we present two enabling optical designs for multiphoton imaging of responses to visual stimuli in mouse retinas expressing calcium indicators. First, we present an imaging solution based on Scanning Line Temporal Focusing (SLITE) for rapidly imaging neuronal activity in vitro. In this design, we scan a temporally focused line rather than a point, increasing the scan speed and reducing the impact of repeated excitation, while maintaining high optical sectioning. Second, we present the first in vivo demonstration of two-photon imaging of RGC activity in the mouse retina. To obtain these cellular resolution recordings we integrated an illumination path into a correction-free imaging system designed using an optical model of the mouse eye. This system can image at multiple depths using an electronically tunable lens integrated into its optical path. The new optical designs presented here overcome a number of outstanding obstacles, allowing the study of rapid calcium- and potentially even voltage-indicator signals both in vitro and in vivo, thereby bringing us a step closer toward distributed monitoring of action potentials.

  3. Spectral Description of Multi-Photon Processes in Quantized Many-Electron Systems Based on a Reduced-Density-Matrix Approach

    NASA Astrophysics Data System (ADS)

    Jacobs, Verne

    2015-05-01

    The frequency-dependent transition rates for multi-photon processes in quantized many-electron systems are evaluated using a reduced-density-matrix approach. A fundamental foundation, based on quantum electrodynamics, is provided for systematic spectral simulations for electromagnetic interactions in quantized many-electron systems, including atomic, molecular, and solid-state systems. A perturbation expansion of the frequency-domain Liouville-space self-energy operator is employed in detailed evaluations of the spectral-line shapes. The self-energy contributions associated with environmental electron-photon and electron-phonon interactions are systematically taken into account. Detailed evaluations have been carried out for the spectral-line widths and shifts in the diagonal-resolvent, lowest order (Born), and short-memory-time (Markov) approximations. Work supported by the Office of Naval Research through the Basic Research Program at The Naval Research Laboratory.

  4. Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

    PubMed

    Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

    2017-02-24

    An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP](mi2004) zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

  5. Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics.

    PubMed

    Bancelin, Stéphane; Decencière, Etienne; Machairas, Vaïa; Albert, Claire; Coradin, Thibaud; Schanne-Klein, Marie-Claire; Aimé, Carole

    2014-09-21

    The assembly of proteins into fibrillar structures is an important process that concerns different biological contexts, including molecular medicine and functional biomaterials. Engineering of hybrid biomaterials can advantageously provide synergetic interactions of the biopolymers with an inorganic component to ensure specific supramolecular organization and dynamics. To this aim, we designed hybrid systems associating collagen and surface-functionalized silica particles and we built a new strategy to investigate fibrillogenesis processes in such multicomponents systems, working at the crossroads of chemistry, physics and mathematics. The self-assembly process was investigated by bimodal multiphoton imaging coupling second harmonic generation (SHG) and 2 photon excited fluorescence (2PEF). The in-depth spatial characterization of the system was further achieved using the three-dimensional analysis of the SHG/2PEF data via mathematical morphology processing. Quantitation of collagen distribution around particles offers strong evidence that the chemically induced confinement of the protein on the silica nanosurfaces has a key influence on the spatial extension of fibrillogenesis. This new approach is unique in the information it can provide on 3D dynamic hybrid systems and may be extended to other associations of fibrillar molecules with optically responsive nano-objects.

  6. Characterization of a Spectral Imaging System

    NASA Astrophysics Data System (ADS)

    Gebhart, Steven C.; Lin, Wei-Chiang; Mahadevan-Jansen, Anita

    2003-07-01

    Complete infiltrating brain tumor margin resection continually eludes neurosurgeons due to inherent limitations of current margin localization techniques. A need exists for an objective, on-site, real-time imaging system which can accurately localize brain tumor margins and therefore be used as a basis for image-guided surgery. Optical biopsy methods are a proven means for successful brain tissue discrimination, indicating promise for spectral imaging to fill such a need. Before testing spectral imaging for surgical guidance, various spectral imaging modalities must be systematically compared to determine the modality most conducive to the clinical setting. A liquid crystal tunable filter spectral imaging system was characterized for field of view, spatial and spectral resolution, and ability to retain spectral features acquired from a clinical single-pixel spectroscopy system. For a 35-mm diameter field of view, the system possessed a spatial resolution of 50 μm in both image dimensions and a spectral resolution which monotonically increased from 10 to 30 nm over the tuning range of the filter. Differences between imaging and single-pixel spectra for location and FWHM of fluorescence peaks from two fluorescent dye targets were summarily less than 3 nm. However, two remediable artifacts were introduced to imaging system spectra during spectral sensitivity correction.

  7. Design and commissioning of a directly coupled in-vivo multiphoton microscope for skin imaging in humans and large animals

    NASA Astrophysics Data System (ADS)

    Mulholland, William J.; Kendall, Mark A.

    2004-02-01

    The application of near infrared multiphoton excitation to the laser-scanning microscope was first conceived by Denk, Strickler and Webb in 1990. Since then, advances in design have seen the multiphoton laser scanning microscope (MPLSM) applied to a wide range of biological research areas, including skin imaging and vaccine delivery. The technique has the attributes of low phototoxicity, high-resolution functional imaging to depths in scattered tissues. These characteristics have encouraged engineers and scientists to develop in-vivo imaging systems. For these applications, laser excitation pulses can be delivered to the sample through optical fibers. Although this solution provides a number of advantages relating to movement and flexibility of the site of interest relative to the laser source, the peak powers that can be delivered down the fiber are limited. We report on the design and commissioning of a directly coupled in-vivo MPM system, optimised for the imaging of epidermal vaccines delivered to a range of biological models and humans. Specifically, we seek to apply the system to visualise in-vivo, the influence of hand-held, helium powered needle-free systems on skin cells. A standard Nikon E600FN microscope, dissected above the optical plane was cantilevered from a vibration isolated table using rigid support arms. The modified microscope was coupled to an infrared optimised Bio-Rad Radiance 2100MP, multiphoton dedicated laser scanning control and image acquisition system. Femtosecond laser pulses were provided by a 10W Verdi pumped Mira Ti:Sapphire laser, from Coherent Inc. The microscope was modified such that the transmission half may be selectively attached for conventional imaging with ex-vivo and cell culture samples, or removed for in-vivo imaging of skin sites on the body of humans and large animals. Optical performance of the system, and aspects of its design and commissioning are discussed in this paper.

  8. Spectral imaging of the human ocular fundus

    NASA Astrophysics Data System (ADS)

    Truitt, Paul Wiley

    Introduction. The objective of this work was to demonstrate a high spectral and spatial resolution fundus imager and to assess its utility in visualizing and characterizing normal anatomical and pathological tissue classes in the human ocular fundus. The ocular fundus (posterior portion of the eye) affords a unique opportunity to directly observe neural and vascular tissue in vivo. Many ocular and systemic diseases manifest changes in the normal fundus anatomy. Current examination techniques are not optimized to detect changes prior to the formation of damaging lesions. Spectral imaging may allow visualization of disease states before the onset of traditional clinical signs. Normal tissue in the eye has distinct spectral characteristics determined by specific structural organization and the presence of specific chemical substances and ocular pigments. Pathological states result in physical and chemical changes to the tissue. Spectral imaging exploits the differences in the spectral characteristics to separate different classes of material. When these spectral properties are combined with the spatial context of the image, improved visualization and detection is possible. Methods. Two independent spectral imaging devices were developed and integrated to a commercially available Zeiss fundus camera. Spectral data were collected in order to characterize the normal anatomical tissue classes and to assess the usefulness of spectral features for improved class discernment. Spectral images were collected for 14 subjects Diabetic Retinopathy were imaged. Mean spectral curves were produced for each class and for each subject. These spectral curves were normalized to remove the contribution from the pigment melanin (the major pigment associated with variation in fundus pigmentation) and modeled with a piece-wise linear function consisting of a DC offset and four slopes. Results. Differences in the shape of the spectral curve exist between macular edema and normal macular and

  9. Optimal spectral windows for microwave diversity imaging

    NASA Technical Reports Server (NTRS)

    Farhat, Nabil H.; Bai, Baocheng

    1991-01-01

    Tomographic microwave diversity imaging is analyzed using linear system theory concepts, and optimal spectral windows for data acquisition are obtained either by considering window position in the spectral domain or by using simulated annealing to find an optimal phase weighting of the object frequency response samples collected over the specified spectral window. This study provides a means of microwave image formation that is applicable under general assumptions. Results of numerical simulations and representative images reconstructed from realistic experimental microwave scattering data are given, demonstrating that the proposed approach is superior to previous image reconstruction methods.

  10. Novel techniques with multiphoton microscopy: Deep-brain imaging with microprisms, neurometabolism of epilepsy, and counterfeit paper money detection

    NASA Astrophysics Data System (ADS)

    Chia, Thomas H.

    Multiphoton microscopy is a laser-scanning fluorescence imaging method with extraordinary potential. We describe three innovative multiphoton microscopy techniques across various disciplines. Traditional in vivo fluorescence microscopy of the mammalian brain has a limited penetration depth (<400 microm). We present a method of imaging 1 mm deep into mouse neocortex by using a glass microprism to relay the excitation and emission light. This technique enables simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution was sufficient to resolve dendritic spines on layer V GFP neurons. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. Multiphoton fluorescence lifetime imaging (FLIM) of NADH reveals information on neurometabolism. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic coenzyme, has a lifetime dependent on enzymatic binding. A novel NADH FLIM algorithm is presented that produces images showing spatially distinct NADH fluorescence lifetimes in mammalian brain slices. This program provides advantages over traditional FLIM processing of multi-component lifetime data. We applied this technique to a GFP-GFAP pilocarpine mouse model of temporal lobe epilepsy. Results indicated significant changes in the neurometabolism of astrocytes and neuropil in the cell and dendritic layers of the hippocampus when compared to control tissue. Data obtained with NADH FLIM were subsequently interpreted based on the abnormal activity reported in epileptic tissue. Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used

  11. Spectral modulation interferometry for quantitative phase imaging

    PubMed Central

    Shang, Ruibo; Chen, Shichao; Li, Chengshuai; Zhu, Yizheng

    2015-01-01

    We propose a spectral-domain interferometric technique, termed spectral modulation interferometry (SMI), and present its application to high-sensitivity, high-speed, and speckle-free quantitative phase imaging. In SMI, one-dimensional complex field of an object is interferometrically modulated onto a broadband spectrum. Full-field phase and intensity images are obtained by scanning along the orthogonal direction. SMI integrates the high sensitivity of spectral-domain interferometry with the high speed of spectral modulation to quantify fast phase dynamics, and its dispersive and confocal nature eliminates laser speckles. The principle and implementation of SMI are discussed. Its performance is evaluated using static and dynamic objects. PMID:25780737

  12. Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging.

    PubMed

    Mancuso, James J; Larson, Adam M; Wensel, Theodore G; Saggau, Peter

    2009-01-01

    The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

  13. Iterative image reconstruction in spectral CT

    NASA Astrophysics Data System (ADS)

    Hernandez, Daniel; Michel, Eric; Kim, Hye S.; Kim, Jae G.; Han, Byung H.; Cho, Min H.; Lee, Soo Y.

    2012-03-01

    Scan time of spectral-CTs is much longer than conventional CTs due to limited number of x-ray photons detectable by photon-counting detectors. However, the spectral pixel information in spectral-CT has much richer information on physiological and pathological status of the tissues than the CT-number in conventional CT, which makes the spectral- CT one of the promising future imaging modalities. One simple way to reduce the scan time in spectral-CT imaging is to reduce the number of views in the acquisition of projection data. But, this may result in poorer SNR and strong streak artifacts which can severely compromise the image quality. In this work, spectral-CT projection data were obtained from a lab-built spectral-CT consisting of a single CdTe photon counting detector, a micro-focus x-ray tube and scan mechanics. For the image reconstruction, we used two iterative image reconstruction methods, the simultaneous iterative reconstruction technique (SIRT) and the total variation minimization based on conjugate gradient method (CG-TV), along with the filtered back-projection (FBP) to compare the image quality. From the imaging of the iodine containing phantoms, we have observed that SIRT and CG-TV are superior to the FBP method in terms of SNR and streak artifacts.

  14. Nonlinear spectral imaging of biological tissues

    NASA Astrophysics Data System (ADS)

    Palero, J. A.

    2007-07-01

    The work presented in this thesis demonstrates live high resolution 3D imaging of tissue in its native state and environment. The nonlinear interaction between focussed femtosecond light pulses and the biological tissue results in the emission of natural autofluorescence and second-harmonic signal. Because biological intrinsic emission is generally very weak and extends from the ultraviolet to the visible spectral range, a broad-spectral range and high sensitivity 3D spectral imaging system is developed. Imaging the spectral characteristics of the biological intrinsic emission reveals the structure and biochemistry of the cells and extra-cellular components. By using different methods in visualizing the spectral images, discrimination between different tissue structures is achieved without the use of any stain or fluorescent label. For instance, RGB real color spectral images of the intrinsic emission of mouse skin tissues show blue cells, green hair follicles, and purple collagen fibers. The color signature of each tissue component is directly related to its characteristic emission spectrum. The results of this study show that skin tissue nonlinear intrinsic emission is mainly due to the autofluorescence of reduced nicotinamide adenine dinucleotide (phosphate), flavins, keratin, melanin, phospholipids, elastin and collagen and nonlinear Raman scattering and second-harmonic generation in Type I collagen. In vivo time-lapse spectral imaging is implemented to study metabolic changes in epidermal cells in tissues. Optical scattering in tissues, a key factor in determining the maximum achievable imaging depth, is also investigated in this work.

  15. Highlight detection and removal from spectral image.

    PubMed

    Koirala, Pesal; Pant, Paras; Hauta-Kasari, Markku; Parkkinen, Jussi

    2011-11-01

    We present a constrained spectral unmixing method to remove highlight from a single spectral image. In the constrained spectral unmixing method, the constraints have been imposed so that all the fractions of diffuse and highlight reflection sum up to 1 and are positive. As a result, the spectra of the diffuse image are always positive. The spectral power distribution (SPD) of the light source has been used as the pure highlight spectrum. The pure diffuse spectrum of the measured spectrum has been chosen from the set of diffuse spectra. The pure diffuse spectrum has a minimum angle among the angles calculated between spectra from a set of diffuse spectra and the measured spectrum projected onto the subspace orthogonal to the SPD of the light source. The set of diffuse spectra has been collected by an automated target generation program from the diffuse part in the image. Constrained energy minimization in a finite impulse response linear filter has been used to detect the highlight and diffuse parts in the image. Results by constrained spectral unmixing have been compared with results by the orthogonal subspace projection (OSP) method [Proceedings of International Conference on Pattern Recognition (2006), pp. 812-815] and probabilistic principal component analysis (PPCA) [Proceedings of the 4th WSEAS International Conference on Signal Processing, Robotics and Automation (2005), paper 15]. Constrained spectral unmixing outperforms OSP and PPCA in the visual assessment of the diffuse results. The highlight removal method by constrained spectral unmixing is suitable for spectral images.

  16. Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination

    PubMed Central

    Therrien, O. D.; Aubé, B.; Pagès, S.; Koninck, P. De; Côté, D.

    2011-01-01

    Wide-field temporal focusing is a novel technique that provides optical sectioning for imaging without the need for beam scanning. However, illuminating over large areas greatly reduces the photon density which limits the technique applicability to small regions, precluding functional imaging of cellular networks. Here we present a strategy that combines beam shaping and temporal focusing of amplified pulses (>1 µJ/pulse) for fast imaging of cells from the central nervous system in acute slices. Multiphoton video-rate imaging over total areas as wide as 4800 µm2 with an optical sectioning under 10 µm at 800 nm is achieved with our setup, leading to imaging of calcium dynamics of multiple cells simultaneously in thick tissue. PMID:21412473

  17. Multiphoton tomography of intratissue tattoo nanoparticles

    NASA Astrophysics Data System (ADS)

    König, Karsten

    2012-02-01

    Most of today's intratissue tattoo pigments are unknown nanoparticles. So far, there was no real control of their use due to the absence of regulations. Some of the tattoo pigments contain carcinogenic amines e.g. azo pigment Red 22. Nowadays, the European Union starts to control the administration of tattoo pigments. There is an interest to obtain information on the intratissue distribution, their interaction with living cells and the extracellular matrix, and the mechanisms behind laser tattoo removal. Multiphoton tomographs are novel biosafety and imaging tools that can provide such information non-invasively and without further labeling. When using the spectral FLIM module, spatially-resolved emission spectra, excitation spectra, and fluorescence lifetimes can pr provided. Multiphoton tomographs are used by all major cosmetic comapanies to test the biosafety of sunscreen nanoparticles.

  18. New applications of Spectral Edge image fusion

    NASA Astrophysics Data System (ADS)

    Hayes, Alex E.; Montagna, Roberto; Finlayson, Graham D.

    2016-05-01

    In this paper, we present new applications of the Spectral Edge image fusion method. The Spectral Edge image fusion algorithm creates a result which combines details from any number of multispectral input images with natural color information from a visible spectrum image. Spectral Edge image fusion is a derivative-based technique, which creates an output fused image with gradients which are an ideal combination of those of the multispectral input images and the input visible color image. This produces both maximum detail and natural colors. We present two new applications of Spectral Edge image fusion. Firstly, we fuse RGB-NIR information from a sensor with a modified Bayer pattern, which captures visible and near-infrared image information on a single CCD. We also present an example of RGB-thermal image fusion, using a thermal camera attached to a smartphone, which captures both visible and low-resolution thermal images. These new results may be useful for computational photography and surveillance applications.

  19. Multiphoton Imaging of Rabbit Cornea Treated with Mitomycin C after Photorefractive Keratectomy

    NASA Astrophysics Data System (ADS)

    Hsueh, Chiu-Mei; Lo, Wen; Wang, Tsung-Jen; Hu, Fung-Rong; Dong, Chen-Yuan

    2007-07-01

    In this work we use multiphoton microscopy to observe the post surgery structure variation of rabbit cornea after photorefractive keratectomy (PRK). In addition, we added mitomycin C (MMC) to the post surgery rabbit cornea in order to investigate the effect of MMC treatment on the postoperative regeneration.

  20. Transverse correlations in multiphoton entanglement

    SciTech Connect

    Wen Jianming; Rubin, Morton H.; Shih Yanhua

    2007-10-15

    We have analyzed the transverse correlation in multiphoton entanglement. The generalization of quantum ghost imaging is extended to the N-photon state. The Klyshko's two-photon advanced-wave picture is generalized to the N-photon case.

  1. High-fidelity spatially resolved multiphoton counting for quantum imaging applications.

    PubMed

    Chrapkiewicz, Radosław; Wasilewski, Wojciech; Banaszek, Konrad

    2014-09-01

    We present a method for spatially resolved multiphoton counting based on an intensified camera with the retrieval of multimode photon statistics fully accounting for nonlinearities in the detection process. The scheme relies on one-time quantum tomographic calibration of the detector. Faithful, high-fidelity reconstruction of single- and two-mode statistics of multiphoton states is demonstrated for coherent states and their statistical mixtures. The results consistently exhibit classical values of the Mandel parameter and the noise reduction factor in contrast to raw statistics of camera photo-events. Detector operation is reliable for illumination levels up to the average of one detected photon per an event area-substantially higher than in previous approaches to characterize quantum statistical properties of light with spatial resolution.

  2. Spectral calibration for convex grating imaging spectrometer

    NASA Astrophysics Data System (ADS)

    Zhou, Jiankang; Chen, Xinhua; Ji, Yiqun; Chen, Yuheng; Shen, Weimin

    2013-12-01

    Spectral calibration of imaging spectrometer plays an important role for acquiring target accurate spectrum. There are two spectral calibration types in essence, the wavelength scanning and characteristic line sampling. Only the calibrated pixel is used for the wavelength scanning methods and he spectral response function (SRF) is constructed by the calibrated pixel itself. The different wavelength can be generated by the monochromator. The SRF is constructed by adjacent pixels of the calibrated one for the characteristic line sampling methods. And the pixels are illuminated by the narrow spectrum line and the center wavelength of the spectral line is exactly known. The calibration result comes from scanning method is precise, but it takes much time and data to deal with. The wavelength scanning method cannot be used in field or space environment. The characteristic line sampling method is simple, but the calibration precision is not easy to confirm. The standard spectroscopic lamp is used to calibrate our manufactured convex grating imaging spectrometer which has Offner concentric structure and can supply high resolution and uniform spectral signal. Gaussian fitting algorithm is used to determine the center position and the Full-Width-Half-Maximum(FWHM)of the characteristic spectrum line. The central wavelengths and FWHMs of spectral pixels are calibrated by cubic polynomial fitting. By setting a fitting error thresh hold and abandoning the maximum deviation point, an optimization calculation is achieved. The integrated calibration experiment equipment for spectral calibration is developed to enhance calibration efficiency. The spectral calibration result comes from spectral lamp method are verified by monochromator wavelength scanning calibration technique. The result shows that spectral calibration uncertainty of FWHM and center wavelength are both less than 0.08nm, or 5.2% of spectral FWHM.

  3. Compact clinical high-NA multiphoton endoscopy

    NASA Astrophysics Data System (ADS)

    Weinigel, Martin; Breunig, Hans Georg; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; König, Karsten

    2012-02-01

    Multiphoton imaging methods are excellent for non-invasive imaging of living tissue without any need of additional contrast agents. The increasing demand for endoscopic techniques has forced the development of multiphoton endoscopes for imaging of areas with reduced accessibility like chronic wounds. Gradient index (GRIN) lenses can miniaturize the bulky distal focusing optics of conventional tomographs to a diameter of less than 1.4 mm and a numerical aperture (NA) of 0.8. We combined a high NA clinical multiphoton endoscope with existing multiphoton tomographs like the DermaInspect® and the MPTflex® to enable the examination of wound healing processes.

  4. Information-efficient spectral imaging sensor

    DOEpatents

    Sweatt, William C.; Gentry, Stephen M.; Boye, Clinton A.; Grotbeck, Carter L.; Stallard, Brian R.; Descour, Michael R.

    2003-01-01

    A programmable optical filter for use in multispectral and hyperspectral imaging. The filter splits the light collected by an optical telescope into two channels for each of the pixels in a row in a scanned image, one channel to handle the positive elements of a spectral basis filter and one for the negative elements of the spectral basis filter. Each channel for each pixel disperses its light into n spectral bins, with the light in each bin being attenuated in accordance with the value of the associated positive or negative element of the spectral basis vector. The spectral basis vector is constructed so that its positive elements emphasize the presence of a target and its negative elements emphasize the presence of the constituents of the background of the imaged scene. The attenuated light in the channels is re-imaged onto separate detectors for each pixel and then the signals from the detectors are combined to give an indication of the presence or not of the target in each pixel of the scanned scene. This system provides for a very efficient optical determination of the presence of the target, as opposed to the very data intensive data manipulations that are required in conventional hyperspectral imaging systems.

  5. From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    PubMed Central

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-01-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy. PMID:27004454

  6. From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    NASA Astrophysics Data System (ADS)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-03-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

  7. Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue.

    PubMed

    Makale, Milan; McElroy, Michele; O'Brien, Peter; Hoffman, Robert M; Guo, Sharon; Bouvet, Michael; Barnes, Leo; Ingulli, Elizabeth; Cheresh, David

    2009-01-01

    We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 microm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 microm. This compares favorably with the 100- to 300 microm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40-microm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.

  8. Multiphoton microscopic imaging of histological sections without hematoxylin and eosin staining differentiates carcinoma in situ lesion from normal oesophagus

    NASA Astrophysics Data System (ADS)

    Chen, Jianxin; Xu, Jian; Kang, Deyong; Xu, Meifang; Zhuo, Shuangmu; Zhu, Xiaoqin; Jiang, Xingshan

    2013-10-01

    Multiphoton microscopy (MPM) has become a powerful, important tool for tissues imaging at the molecular level. In this paper, this technique was extended to histological investigations, differentiating carcinoma in situ (CIS) lesion from normal oesophagus by imaging histological sections without hematoxylin and eosin (H&E) staining. The results show that the histology procedures of dehydration, paraffin embedding, and de-paraffinizing highlighted two photon excited fluorescence of cytoplasm and nucleolus of epithelial cell and collagen in stroma. MPM has the ability to identify the characteristics of CIS lesion including changes of squamous cells and full epithelium, identification of basement membrane, especially prominent nucleolus. The studies described here show that MPM has the potential for future retrospective studies of tumor staging by employing on histological section specimens without H&E staining.

  9. Multi Spectral Fluorescence Imager (MSFI)

    NASA Technical Reports Server (NTRS)

    Caron, Allison

    2016-01-01

    Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.

  10. SPECTRAL SMILE CORRECTION IN CRISM HYPERSPECTRAL IMAGES

    NASA Astrophysics Data System (ADS)

    Ceamanos, X.; Doute, S.

    2009-12-01

    The Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) is affected by a common artifact in "push-broom" sensors, the so-called "spectral smile". As a consequence, both central wavelength and spectral width of the spectral response vary along the across-track dimension, thus giving rise to a shifting and smoothing of spectra (see Fig. 1 (left)). In fact, both effects are greater for spectra on the edges, while they are minimum for data acquired by central detectors, the so-called "sweet spot". The prior artifacts become particularly critical for Martian observations which contain steep spectra such as CO2 ice-rich polar images. Fig. 1 (right) shows the horizontal brightness gradient which appears in every band corresponding to a steep portion of spectra. The correction of CRISM spectral smile is addressed using a two-step method which aims at modifying data sensibly in order to mimic the optimal CRISM response. First, all spectra, which are previously interpolated by cubic splines, are resampled to the "sweet spot" wavelengths in order to overcome the spectra shift. Secondly, the non-uniform spectral width is overcome by mimicking an increase of spectral resolution thanks to a spectral sharpening. In order to minimize noise, only bands particularly suffering from smile are selected. First, bands corresponding to the outliers of the Minimum Noise Transformation (MNF) eigenvector, which corresponds to the MNF band related to smile (MNF-smile), are selected. Then, a spectral neighborhood Θi, which takes into account the local spectral convexity or concavity, is defined for every selected band in order to maximize spectral shape preservation. The proposed sharpening technique takes into account both the instrument parameters and the observed spectra. First, every reflectance value belonging to a Θi is reevaluated by a sharpening which depends on a ratio of the spectral width of the current detector and the "sweet spot" one. Then, the optimal degree of

  11. Spectral clustering algorithms for ultrasound image segmentation.

    PubMed

    Archip, Neculai; Rohling, Robert; Cooperberg, Peter; Tahmasebpour, Hamid; Warfield, Simon K

    2005-01-01

    Image segmentation algorithms derived from spectral clustering analysis rely on the eigenvectors of the Laplacian of a weighted graph obtained from the image. The NCut criterion was previously used for image segmentation in supervised manner. We derive a new strategy for unsupervised image segmentation. This article describes an initial investigation to determine the suitability of such segmentation techniques for ultrasound images. The extension of the NCut technique to the unsupervised clustering is first described. The novel segmentation algorithm is then performed on simulated ultrasound images. Tests are also performed on abdominal and fetal images with the segmentation results compared to manual segmentation. Comparisons with the classical NCut algorithm are also presented. Finally, segmentation results on other types of medical images are shown.

  12. Multiphoton imaging to distinguish grana and starch inside an intact leaf

    NASA Astrophysics Data System (ADS)

    Chen, Mei-Yu; Zhuo, Guan-Yu; Chen, Po-Fu; Wu, Pei-Chun; Liu, Tzu-Ming; Chu, Shi-Wei

    2013-02-01

    We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. Grana and starch are the major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two photon fluorescence (2PF) and second harmonic generation (SHG) signals. Consequently, SHG is found on both grana and starch while 2PF from chlorophyll indicates the identity of grana.

  13. Multicolor multiphoton microscopy based on a nanosecond supercontinuum laser source.

    PubMed

    Lefort, Claire; O'Connor, Rodney P; Blanquet, Véronique; Magnol, Laetitia; Kano, Hideaki; Tombelaine, Vincent; Lévêque, Philippe; Couderc, Vincent; Leproux, Philippe

    2016-07-01

    Multicolor multiphoton microscopy is experimentally demonstrated for the first time on a spectral bandwidth of excitation of 300 nm (full width half maximum) thanks to the implementation a nanosecond supercontinuum (SC) source compact and simple with a low repetition rate. The interest of such a wide spectral bandwidth, never demonstrated until now, is highlighted in vivo: images of glioma tumor cells stably expressing eGFP grafted on the brain of a mouse and its blood vessels network labelled with Texas Red(®) are obtained. These two fluorophores have a spectral bandwidth covering the whole 300 nm available. In parallel, a similar image quality is obtained on a sample of mouse muscle in vitro when excited with this nanosecond SC source or with a classical high rate, femtosecond and quasi monochromatic laser. This opens the way for (i) a simple and very complete biological characterization never performed to date with multiphoton processes, (ii) multiple means of contrast in nonlinear imaging allowed by the use of numerous fluorophores and (iii) other multiphoton processes like three-photon ones.

  14. Remote Sensing of Landscapes with Spectral Images

    NASA Astrophysics Data System (ADS)

    Adams, John B.; Gillespie, Alan R.

    2006-05-01

    Remote Sensing of Landscapes with Spectral Images describes how to process and interpret spectral images using physical models to bridge the gap between the engineering and theoretical sides of remote-sensing and the world that we encounter when we venture outdoors. The emphasis is on the practical use of images rather than on theory and mathematical derivations. Examples are drawn from a variety of landscapes and interpretations are tested against the reality seen on the ground. The reader is led through analysis of real images (using figures and explanations); the examples are chosen to illustrate important aspects of the analytic framework. This textbook will form a valuable reference for graduate students and professionals in a variety of disciplines including ecology, forestry, geology, geography, urban planning, archeology and civil engineering. It is supplemented by a web-site hosting digital color versions of figures in the book as well as ancillary images (www.cambridge.org/9780521662214). Presents a coherent view of practical remote sensing, leading from imaging and field work to the generation of useful thematic maps Explains how to apply physical models to help interpret spectral images Supplemented by a website hosting digital colour versions of figures in the book, as well as additional colour figures

  15. Clinical multiphoton and CARS microscopy

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Weinigel, M.; Darvin, M. E.; Lademann, J.; König, K.

    2012-03-01

    We report on clinical CARS imaging of human skin in vivo with the certified hybrid multiphoton tomograph CARSDermaInspect. The CARS-DermaInspect provides simultaneous imaging of non-fluorescent intradermal lipid and water as well as imaging of two-photon excited fluorescence from intrinsic molecules. Two different excitation schemes for CARS imaging have been realized: In the first setup, a combination of fs oscillator and optical parametric oscillator provided fs-CARS pump and Stokes pulses, respectively. In the second setup a fs oscillator was combined with a photonic crystal fiber which provided a broadband spectrum. A spectral range out of the broadband-spectrum was selected and used for CARS excitation in combination with the residual fs-oscillator output. In both setups, in addition to CARS, single-beam excitation was used for imaging of two-photon excited fluorescence and second harmonic generation signals. Both CARS-excitation systems were successfully used for imaging of lipids inside the skin in vivo.

  16. Miniaturized spectral imager for Aalto-1 nanosatellite

    NASA Astrophysics Data System (ADS)

    Mannila, Rami; Näsilä, Antti; Praks, Jaan; Saari, Heikki; Antila, Jarkko

    2011-11-01

    The Aalto-1 is a 3U-cubesat project coordinated by Aalto University. The satellite, Aalto-1, will be mainly built by students as project assignments and thesis works. VTT Technical Research Centre of Finland will develop the main Earth observation payload, a miniaturized spectral imager, for the satellite. It is a novel highly miniaturized tunable filter type spectral imager. Mass of the spectral imager will be less than 400 grams, and dimensions will be approximately 80 mm x 80 mm x 45 mm. The spectral imager is based on a tunable Fabry-Pérot interferometer (FPI) accompanied by an RGB CMOS image sensor. The FPI consists of two highly reflective surfaces separated by a tunable air gap and it is based either on a microelectromechanical (MEMS) or piezo-actuated structure. The MEMS FPI is a monolithic device, i.e. it is made entirely on one substrate in a batch process, without assembling separate pieces together. The gap is adjusted by moving the upper mirror with electrostatic force. Benefits of the MEMS FPI are low mass and small size. However, large aperture (2-10 mm) MEMS FPIs are currently under development, thus it is not yet known if their performance is adequate. The piezo-actuated FPI uses three piezo-actuators and is controlled in a closed capacitive feedback loop. The drawback of the piezo-actuated FPI is its higher mass. However, it has a large aperture which enables a shorter exposure times. Selection of the FPI type will be done after thorough evaluation. Depending on the selected FPI type, the spectral resolution of the imager will be 5 - 10 nm at full width at half maximum and it will operate in the visible and/or near infrared range.

  17. Wavelength dependent photoelectron circular dichroism of limonene studied by femtosecond multiphoton laser ionization and electron-ion coincidence imaging

    NASA Astrophysics Data System (ADS)

    Rafiee Fanood, Mohammad M.; Janssen, Maurice H. M.; Powis, Ivan

    2016-09-01

    Enantiomers of the monoterpene limonene have been investigated by (2 + 1) resonance enhanced multiphoton ionization and photoelectron circular dichroism employing tuneable, circularly polarized femtosecond laser pulses. Electron imaging detection provides 3D momentum measurement while electron-ion coincidence detection can be used to mass-tag individual electrons. Additional filtering, by accepting only parent ion tagged electrons, can be then used to provide discrimination against higher energy dissociative ionization mechanisms where more than three photons are absorbed to better delineate the two photon resonant, one photon ionization pathway. The promotion of different vibrational levels and, tentatively, different electronic ion core configurations in the intermediate Rydberg states can be achieved with different laser excitation wavelengths (420 nm, 412 nm, and 392 nm), in turn producing different state distributions in the resulting cations. Strong chiral asymmetries in the lab frame photoelectron angular distributions are quantified, and a comparison made with a single photon (synchrotron radiation) measurement at an equivalent photon energy.

  18. In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy

    SciTech Connect

    Zhuo, Shuangmu E-mail: hanry-yu@nuhs.edu.sg; Yan, Jie; Kang, Yuzhan; Peng, Qiwen; and others

    2014-07-14

    Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.

  19. The design of double electrostatic-lens optics for resonance enhanced multiphoton ionization and photoelectron imaging experiments

    SciTech Connect

    Qu, Zehua; Li, Chunsheng; Qin, Zhengbo E-mail: xfzheng@mail.ahnu.edu.cn; Zheng, Xianfeng E-mail: xfzheng@mail.ahnu.edu.cn; Yao, Guanxin; Zhang, Xianyi; Cui, Zhifeng

    2015-06-15

    Compared to single ion/electron-optics for velocity-map imaging, a double-focusing lens assembly designed not only allows for mapping velocity imaging of photoelectrons but also allows for investigating the vibrational structure of the intermediate states of neutral species in resonance enhanced multiphoton ionization (REMPI) spectra. In this presentation, in order to record REMPI and photoelectron spectra separately, we have constructed a compact photoelectron velocity-map imaging (VMI) apparatus combined with an opposite linear Wiley-Mclaren time-of-flight mass spectrometer (TOFMS). A mass resolution (m/Δm) of ∼1300 for TOFMS and electron energy resolution (ΔE/E) of 2.4% for VMI have been achieved upon three-photon ionization of Xe atom at 258.00 nm laser wavelength. As a benchmark, in combination of one-color (1 + 1) REMPI and photoelectron imaging of benzene via 6{sup 1} and 6{sup 1}1{sup 1} vibronic levels in the S{sub 1} state, the vibrational structures of the cation and photoelectron angular anisotropy are unraveled. In addition, two-color (1 + 1′) REMPI and photoelectron imaging of aniline was used to complete the accurate measurement of ionization potential (62 271 ± 3 cm{sup −1}). The results suggest that the apparatus is a powerful tool for studying photoionization dynamics in the photoelectron imaging using vibrational-state selected excitation to the intermediate states of neutrals based on REMPI technique.

  20. The design of double electrostatic-lens optics for resonance enhanced multiphoton ionization and photoelectron imaging experiments

    NASA Astrophysics Data System (ADS)

    Qu, Zehua; Li, Chunsheng; Qin, Zhengbo; Zheng, Xianfeng; Yao, Guanxin; Zhang, Xianyi; Cui, Zhifeng

    2015-06-01

    Compared to single ion/electron-optics for velocity-map imaging, a double-focusing lens assembly designed not only allows for mapping velocity imaging of photoelectrons but also allows for investigating the vibrational structure of the intermediate states of neutral species in resonance enhanced multiphoton ionization (REMPI) spectra. In this presentation, in order to record REMPI and photoelectron spectra separately, we have constructed a compact photoelectron velocity-map imaging (VMI) apparatus combined with an opposite linear Wiley-Mclaren time-of-flight mass spectrometer (TOFMS). A mass resolution (m/Δm) of ˜1300 for TOFMS and electron energy resolution (ΔE/E) of 2.4% for VMI have been achieved upon three-photon ionization of Xe atom at 258.00 nm laser wavelength. As a benchmark, in combination of one-color (1 + 1) REMPI and photoelectron imaging of benzene via 61 and 6111 vibronic levels in the S1 state, the vibrational structures of the cation and photoelectron angular anisotropy are unraveled. In addition, two-color (1 + 1') REMPI and photoelectron imaging of aniline was used to complete the accurate measurement of ionization potential (62 271 ± 3 cm-1). The results suggest that the apparatus is a powerful tool for studying photoionization dynamics in the photoelectron imaging using vibrational-state selected excitation to the intermediate states of neutrals based on REMPI technique.

  1. Analysis of microparticle penetration into human and porcine skin: non-invasive imaging with multiphoton excitation microscopy

    NASA Astrophysics Data System (ADS)

    Mulholland, William J.; Kendall, Mark A.; Bellhouse, Brian J.; White, Nick

    2002-06-01

    At the University of Oxford and PowderJect Pharmaceuticals plc, a unique form of needle-free injection technology has been developed. Powdered vaccines and drugs in micro-particle form are accelerated in a high-speed gas flow to sufficient velocity to enter the skin, subsequently achieving a pharmaceutical effect. To optimize the delivery of vaccines and drugs with this method a detailed understanding of the interactive processes that occur between the microparticles and the skin is necessary. Investigations to date of micro-particle delivery into excised human and animal tissue have involved image analyses of histology sections. In the present study, a series of investigations were conducted on excised human and porcine skin using the technique of Multi-Photon fluorescence excitation Microscopy (MPM) to image particles and skin structures post-penetration. Micro-particles of various size and composition were imaged with infrared laser excitation. Three-dimensional images of stratum corneum and epidermal cell deformation due to micro-particle penetration were obtained. Measurements of micro-particle penetration depth taken from z-scan image stacks were used to successfully quantify micro-particle distribution within the skin, without invasively disrupting the skin target. This study has shown that MPM has great potential for the non-invasive imaging of particle skin interactive processes that occur with the transdermal delivery of powdered micro-particle vaccines and drugs.

  2. Multi-spectral imaging of oxygen saturation

    NASA Astrophysics Data System (ADS)

    Savelieva, Tatiana A.; Stratonnikov, Aleksander A.; Loschenov, Victor B.

    2008-06-01

    The system of multi-spectral imaging of oxygen saturation is an instrument that can record both spectral and spatial information about a sample. In this project, the spectral imaging technique is used for monitoring of oxygen saturation of hemoglobin in human tissues. This system can be used for monitoring spatial distribution of oxygen saturation in photodynamic therapy, surgery or sports medicine. Diffuse reflectance spectroscopy in the visible range is an effective and extensively used technique for the non-invasive study and characterization of various biological tissues. In this article, a short review of modeling techniques being currently in use for diffuse reflection from semi-infinite turbid media is presented. A simple and practical model for use with a real-time imaging system is proposed. This model is based on linear approximation of the dependence of the diffuse reflectance coefficient on relation between absorbance and reduced scattering coefficient. This dependence was obtained with the Monte Carlo simulation of photon propagation in turbid media. Spectra of the oxygenated and deoxygenated forms of hemoglobin differ mostly in the red area (520 - 600 nm) and have several characteristic points there. Thus four band-pass filters were used for multi-spectral imaging. After having measured the reflectance, the data obtained are used for fitting the concentration of oxygenated and free hemoglobin, and hemoglobin oxygen saturation.

  3. Tomographic spectral imaging: analysis of localized corrosion.

    SciTech Connect

    Michael, Joseph Richard; Kotula, Paul Gabriel; Keenan, Michael Robert

    2005-02-01

    Microanalysis is typically performed to analyze the near surface of materials. There are many instances where chemical information about the third spatial dimension is essential to the solution of materials analyses. The majority of 3D analyses however focus on limited spectral acquisition and/or analysis. For truly comprehensive 3D chemical characterization, 4D spectral images (a complete spectrum from each volume element of a region of a specimen) are needed. Furthermore, a robust statistical method is needed to extract the maximum amount of chemical information from that extremely large amount of data. In this paper, an example of the acquisition and multivariate statistical analysis of 4D (3-spatial and 1-spectral dimension) x-ray spectral images is described. The method of utilizing a single- or dual-beam FIB (w/o or w/SEM) to get at 3D chemistry has been described by others with respect to secondary-ion mass spectrometry. The basic methodology described in those works has been modified for comprehensive x-ray microanalysis in a dual-beam FIB/SEM (FEI Co. DB-235). In brief, the FIB is used to serially section a site-specific region of a sample and then the electron beam is rastered over the exposed surfaces with x-ray spectral images being acquired at each section. All this is performed without rotating or tilting the specimen between FIB cutting and SEM imaging/x-ray spectral image acquisition. The resultant 4D spectral image is then unfolded (number of volume elements by number of channels) and subjected to the same multivariate curve resolution (MCR) approach that has proven successful for the analysis of lower-dimension x-ray spectral images. The TSI data sets can be in excess of 4Gbytes. This problem has been overcome (for now) and images up to 6Gbytes have been analyzed in this work. The method for analyzing such large spectral images will be described in this presentation. A comprehensive 3D chemical analysis was performed on several corrosion specimens

  4. Distinguishing human normal or cancerous esophagus tissue ex vivo using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liu, N. R.; Chen, G. N.; Wu, S. S.; Chen, R.

    2014-02-01

    Application of multiphoton microscopy (MPM) to clinical cancer research has greatly developed over the last few years. In this paper, we mainly focus on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) for investigating esophageal cancer. We chiefly discuss the SHG/TPEF image and spectral characteristics of normal and cancerous esophagus submucosa with the combined multi-channel imaging mode and Lambda mode of a multiphoton microscope (LSM 510 META). Great differences can be detected, such as collagen content and morphology, glandular-shaped cancer cells, TPEF/SHG intensity ratio, and so on, which demonstrate that the multiphoton imaging technique has the potential ability for minimally-invasive early cancer diagnosis.

  5. Influence of Vacuum Cooling on Escherichia coli O157:H7 Infiltration in Fresh Leafy Greens via a Multiphoton-Imaging Approach.

    PubMed

    Vonasek, Erica; Nitin, Nitin

    2015-10-16

    Microbial pathogen infiltration in fresh leafy greens is a significant food safety risk factor. In various postharvest operations, vacuum cooling is a critical process for maintaining the quality of fresh produce. The overall goal of this study was to evaluate the risk of vacuum cooling-induced infiltration of Escherichia coli O157:H7 into lettuce using multiphoton microscopy. Multiphoton imaging was chosen as the method to locate E. coli O157:H7 within an intact lettuce leaf due to its high spatial resolution, low background fluorescence, and near-infrared (NIR) excitation source compared to those of conventional confocal microscopy. The variables vacuum cooling, surface moisture, and leaf side were evaluated in a three-way factorial study with E. coli O157:H7 on lettuce. A total of 188 image stacks were collected. The images were analyzed for E. coli O157:H7 association with stomata and E. coli O157:H7 infiltration. The quantitative imaging data were statistically analyzed using analysis of variance (ANOVA). The results indicate that the low-moisture condition led to an increased risk of microbial association with stomata (P < 0.05). Additionally, the interaction between vacuum cooling levels and moisture levels led to an increased risk of infiltration (P < 0.05). This study also demonstrates the potential of multiphoton imaging for improving sensitivity and resolution of imaging-based measurements of microbial interactions with intact leaf structures, including infiltration.

  6. Analysis for simplified optics coma effection on spectral image inversion of coded aperture spectral imager

    NASA Astrophysics Data System (ADS)

    Liu, Yangyang; Lv, Qunbo; Li, Weiyan; Xiangli, Bin

    2015-09-01

    As a novel spectrum imaging technology was developed recent years, push-broom coded aperture spectral imaging (PCASI) has the advantages of high throughput, high SNR, high stability etc. This coded aperture spectral imaging utilizes fixed code templates and push-broom mode, which can realize the high-precision reconstruction of spatial and spectral information. But during optical lens designing, manufacturing and debugging, it is inevitably exist some minor coma errors. Even minor coma errors can reduce image quality. In this paper, we simulated the system optical coma error's influence to the quality of reconstructed image, analyzed the variant of the coded aperture in different optical coma effect, then proposed an accurate curve of image quality and optical coma quality in 255×255 size code template, which provide important references for design and development of push-broom coded aperture spectrometer.

  7. Study of image motion compensation in spectral imaging system

    NASA Astrophysics Data System (ADS)

    Li, Zhijun; Chen, Xing Long

    2016-10-01

    In the spectral imaging system, random jitter and posture change of the aircraft generated random image motion, and flight of aircraft caused forward image motion. Both of image motion can cause image blur in a longer exposure time, which need for image motion compensation. Due to limited field of view of the optical system, limited size and weight, a stable FSM (Fast Steering Mirror) was used for random image motion compensation and a compensation FSM was used for forward image motion compensation. In the random image motion compensation, inertial sensors were used for measuring the random jitter and the posture change of the aircraft. As the advantages and disadvantages for the gyroscope and inclinometer, we used data fusion of the two sensors to complementary advantages with closed-loop mode filter data based on the frequency domain. In this way, we got high linearity, little drift, high bandwidth and little electrical noise inertial measurement sensors. On the other hand, the motion of the compensation mirror was broken down to the amount of displacement within the time required for each interrupt movement. Under strict timing control, macro forward image motion compensation was realized in the exposure time. The above image motion compensation methods were applied to actual spectral imaging systems, aerial experiment results show that image motion compensation obtained good results and met the remaining image motion compensation image error was not more than 1/3 pixel.

  8. Imaging interactions between the immune and cardiovascular systems in vivo by multiphoton microscopy.

    PubMed

    Millington, Owain R; Brewer, James M; Garside, Paul; Maffia, Pasquale

    2010-01-01

    Several recent studies in immunology have used multiphoton laser-scanning microscopy to visualise the induction of an immune response in real time in vivo. These experiments are illuminating the cellular and molecular interactions involved in the induction, maintenance and regulation of immune responses. Similar approaches are being applied in cardiovascular research where there is an increasing body of evidence to support a significant role for the adaptive immune system in vascular disease. As such, we have begun to dissect the role of T lymphocytes in atherosclerosis in real time in vivo. Here, we provide step-by-step guides to the various stages involved in visualising the migration of T cells within a lymph node and their infiltration into inflamed tissues such as atherosclerotic arteries. These methods provide an insight into the mechanisms involved in the activation and function of immune cells in vivo.

  9. In-vivo imaging of psoriatic lesions with polarization multispectral dermoscopy and multiphoton microscopy

    PubMed Central

    Kapsokalyvas, Dimitrios; Cicchi, Riccardo; Bruscino, Nicola; Alfieri, Domenico; Prignano, Francesca; Massi, Daniela; Lotti, Torello; Pavone, Francesco S.

    2014-01-01

    Psoriasis is a skin autoimmune disease characterized by hyperkeratosis, hyperproliferation of the epidermis and dilatation of dermal papillary blood vessels. Healthy skin (5 volunteers) and psoriatic lesions (3 patients) were visualized in vivo, with high contrast and resolution, with a Polarization Multispectral Dermoscope and a Multiphoton Microscope. Psoriatic features were identified and quantified. The effective diameter of the superficial blood vessels was measured at 35.2 ± 7.2 μm and the elongated dermal papillae had an effective diameter of 64.2 ± 22.6 μm. The methodologies developed could be employed for quantitative diagnostic purposes and furthermore serve as a monitoring method of the effect of personalized treatments. PMID:25071974

  10. WAVELENGTH AND ALIGNMENT TESTS FOR CONFOCAL SPECTRAL IMAGING SYSTEMS

    EPA Science Inventory

    Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference s...

  11. CALIBRATION AND VALIDATION OF CONFOCAL SPECTRAL IMAGING SYSTEMS

    EPA Science Inventory

    Confocal spectral imaging (CSI) microscope systems now on the market can perform spectral characterization of biological specimens containing fluorescent proteins, labels or dyes. Some CSI have been found to present inconsistent spectral characterizations within a particular syst...

  12. Quantification of Cy-5 siRNA signal in the intra-vital multi-photon microscopy images.

    PubMed

    Chen, Antong; Dogdas, Belma; Mehta, Saurin; Haskell, Kathleen; Ng, Bruce; Keough, Ed; Howell, Bonnie; Meacham, D Adam; Aslamkhan, Amy G; Davide, Joseph; Stanton, Matthew; Bagchi, Ansuman; Sepp-Lorenzino, Laura; Tao, Weikang

    2012-01-01

    Transgenic mice with Tie2- green fluorescent protein (GFP) are used as a model to study the kinetic distribution of the Cy5-siRNA delivered by lipid nanoparticles (LNP) into the liver. After the mouse is injected with the LNP, it undergoes a procedure of intra-vital multi-photon microscopy imaging over a period of two hours, during which the process for the nanoparticle to diffuse into the hepatocytes from the vasculature system is monitored. Since the images are obtained in-vivo, the quantification of Cy5 kinetics suffers from the moving field of view (FOV). A method is proposed to register the sequence of images through template matching. Based on the semi-automatic segmentations of the vessels in the common FOV, the registered images are segmented into three regions of interest (ROI) in which the Cy5 signals are quantified. Computation of the percentage signal strength in the ROIs over time allows for the analysis of the diffusion of Cy5-siRNA into the hepatocytes, and helps demonstrate the effectiveness of the Cy5-siRNA delivery vehicle.

  13. The layered resolved microstructure and spectroscopy of mouse oral mucosa using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Jiang, Xingshan; Xie, Shusen; Chen, Rong; Cao, Ning; Zou, Qilian; Xiong, Shuyuan

    2007-08-01

    The layered-resolved microstructure and spectroscopy of mouse oral mucosa are obtained using a combination of multiphoton imaging and spectral analysis with different excitation wavelengths. In the keratinizing layer, the keratinocytes microstructure can be characterized and the keratinizing thickness can be measured. The keratin fluorescence signal can be further characterized by emission maxima at 510 nm. In the epithelium, the cellular microstructure can be quantitatively visualized with depth and the epithelium thickness can be determined by multiphoton imaging excited at 730 nm. The study also shows that the epithelial spectra excited at 810 nm, showing a combination of NADH and FAD fluorescence, can be used for the estimation of the metabolic state in epithelium. Interestingly, a second-harmonic generation (SHG) signal from DNA was observed for the first time within the epithelial layer in backscattering geometry and provides the possibility of analyzing the chromatin structure. In the stroma, the combination of multiphoton imaging and spectral analysis excited at 850 nm in tandem can obtain quantitative information regarding the biomorphology and biochemistry of stroma. Specifically, the microstructure of collagen, minor salivary glands and elastic fibers, and the optical property of the stroma can be quantitatively displayed. Overall, these results suggest that the combination of multiphoton imaging and spectral analysis with different excitation wavelengths has the potential to provide important and comprehensive information for early diagnosis of oral cancer.

  14. The spectral imaging facility: Setup characterization

    SciTech Connect

    De Angelis, Simone De Sanctis, Maria Cristina; Manzari, Paola Olga; Ammannito, Eleonora; Di Iorio, Tatiana; Liberati, Fabrizio; Tarchi, Fabio; Dami, Michele; Olivieri, Monica; Pompei, Carlo; Mugnuolo, Raffaele

    2015-09-15

    The SPectral IMager (SPIM) facility is a laboratory visible infrared spectrometer developed to support space borne observations of rocky bodies of the solar system. Currently, this laboratory setup is used to support the DAWN mission, which is in its journey towards the asteroid 1-Ceres, and to support the 2018 Exo-Mars mission in the spectral investigation of the Martian subsurface. The main part of this setup is an imaging spectrometer that is a spare of the DAWN visible infrared spectrometer. The spectrometer has been assembled and calibrated at Selex ES and then installed in the facility developed at the INAF-IAPS laboratory in Rome. The goal of SPIM is to collect data to build spectral libraries for the interpretation of the space borne and in situ hyperspectral measurements of planetary materials. Given its very high spatial resolution combined with the imaging capability, this instrument can also help in the detailed study of minerals and rocks. In this paper, the instrument setup is first described, and then a series of test measurements, aimed to the characterization of the main subsystems, are reported. In particular, laboratory tests have been performed concerning (i) the radiation sources, (ii) the reference targets, and (iii) linearity of detector response; the instrumental imaging artifacts have also been investigated.

  15. Automated eXpert Spectral Image Analysis

    SciTech Connect

    Keenan, Michael R.

    2003-11-25

    AXSIA performs automated factor analysis of hyperspectral images. In such images, a complete spectrum is collected an each point in a 1-, 2- or 3- dimensional spatial array. One of the remaining obstacles to adopting these techniques for routine use is the difficulty of reducing the vast quantities of raw spectral data to meaningful information. Multivariate factor analysis techniques have proven effective for extracting the essential information from high dimensional data sets into a limted number of factors that describe the spectral characteristics and spatial distributions of the pure components comprising the sample. AXSIA provides tools to estimate different types of factor models including Singular Value Decomposition (SVD), Principal Component Analysis (PCA), PCA with factor rotation, and Alternating Least Squares-based Multivariate Curve Resolution (MCR-ALS). As part of the analysis process, AXSIA can automatically estimate the number of pure components that comprise the data and can scale the data to account for Poisson noise. The data analysis methods are fundamentally based on eigenanalysis of the data crossproduct matrix coupled with orthogonal eigenvector rotation and constrained alternating least squares refinement. A novel method for automatically determining the number of significant components, which is based on the eigenvalues of the crossproduct matrix, has also been devised and implemented. The data can be compressed spectrally via PCA and spatially through wavelet transforms, and algorithms have been developed that perform factor analysis in the transform domain while retaining full spatial and spectral resolution in the final result. These latter innovations enable the analysis of larger-than core-memory spectrum-images. AXSIA was designed to perform automated chemical phase analysis of spectrum-images acquired by a variety of chemical imaging techniques. Successful applications include Energy Dispersive X-ray Spectroscopy, X-ray Fluorescence

  16. "Conventional" CT images from spectral measurements

    NASA Astrophysics Data System (ADS)

    Rajbhandary, Paurakh L.; Pelc, Norbert J.

    2016-03-01

    Spectral imaging systems need to be able to produce "conventional" images, and it's been shown that systems with energy discriminating detectors can achieve higher CNR than conventional systems by optimal weighting. Combining measured data in energy bins (EBs) and also combining basis material images have previously been proposed, but there are no studies systematically comparing the two methods. In this paper, we analytically evaluate the two methods for systems with ideal photon counting detectors using CNR and beam hardening (BH) artifact as metrics. For a 120-kVp polychromatic simulations of a water phantom with low contrast inserts, the difference of the optimal CNR between the two methods for the studied phantom is within 2%. For a polychromatic spectrum, beam-hardening artifacts are noticeable in EB weighted images (BH artifact of 3.8% for 8 EB and 6.9% for 2 EB), while weighted basis material images are free of such artifacts.

  17. Detection of latent fingerprints by ultraviolet spectral imaging

    NASA Astrophysics Data System (ADS)

    Huang, Wei; Xu, Xiaojing; Wang, Guiqiang

    2013-12-01

    Spectral imaging technology research is becoming more popular in the field of forensic science. Ultraviolet spectral imaging technology is an especial part of the full spectrum of imaging technology. This paper finished the experiment contents of the ultraviolet spectrum imaging method and image acquisition system based on ultraviolet spectral imaging technology. Ultraviolet spectral imaging experiments explores a wide variety of ultraviolet reflectance spectra of the object material curve and its ultraviolet spectrum of imaging modalities, can not only gives a reference for choosing ultraviolet wavelength to show the object surface potential traces of substances, but also gives important data for the ultraviolet spectrum of imaging technology development.

  18. Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique

    NASA Astrophysics Data System (ADS)

    Lin, Jian; Lu, Fake; Zheng, Wei; Xu, Shuoyu; Tai, Dean; Yu, Hanry; Huang, Zhiwei

    2011-11-01

    We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.

  19. Multiphoton laser tomography and fluorescence lifetime imaging of basal cell carcinoma: morphologic features for non-invasive diagnostics.

    PubMed

    Seidenari, Stefania; Arginelli, Federica; Dunsby, Christopher; French, Paul; König, Karsten; Magnoni, Cristina; Manfredini, Marco; Talbot, Clifford; Ponti, Giovanni

    2012-11-01

    Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus 'other lesions' was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of 'other lesion' extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.

  20. [Microscopic infrared spectral imaging of oily core].

    PubMed

    Huang, Qiao-Song; Yu, Zhao-Xian; Li, Jing; Chen, Chen

    2009-02-01

    In the present paper, the authors examined some oily core by microscopic infrared spectral imaging methods. Those methods can be classified in three modes, referred to as "transmission mode", "reflection mode" and "attenuated total reflection (ATR) mode". The observed oily core samples belong to siltstone. The samples were made of quartz (-20%), feldspar(-50%) and other rock (igneous rock 25%, metamorphic rocks 1%, sedimentary rock 4%); a little recrystallized calcite (-1%) was in the pore, and the argillaceous matter was distributed along the edge of a pore. The experimental work has been accomplished using SHIMADZU Model IRPrestige-21 Fourier transform infrared spectrophotometer plus AIM8800 infrared microscope. For IRPrestige-21, the spectral range is 7 800-350 cm(-1) spectral resolution is 1 cm(-1), and AIM8800 microscope with motorized stages has a resolution of 1 micrometer. The experiment was preformed at room temperature. In "transmission mode" infrared spectral imaging method, the spectral range was limited in wavenumbers greater than 2 000 cm(-1) because the base glass piece has strong light absorption. In contrast with "transmission mode", in "attenuated total reflection (ATR) mode", the depth of penetration into sample is very small (1-2 micrometer), then the absorbance value has nothing to do with base glass piece light absorption. In microscopic infrared transmission spectra, the experimental result shows that there are some strong absorption peaks at 2 866, 2 928, 3 618 and 2 515 cm(-1) respectively. The former two peaks correspond to methyl(methylene) symmetrical and unsymmetrical stretch vibration mode, respectively. The latter two peaks correspond to hydroxyl-stretch vibration mode and S-H, P-H chemical bond stretch vibration mode, respectively. In microscopic longwave infrared ATR spectra, there are other stronger absorption peaks at 1 400, 1 038 and 783 cm(i1)respectively, corresponding to methyl(methylene) widing vibration mode and optical mode

  1. CONSTRAINED SPECTRAL CLUSTERING FOR IMAGE SEGMENTATION

    PubMed Central

    Sourati, Jamshid; Brooks, Dana H.; Dy, Jennifer G.; Erdogmus, Deniz

    2013-01-01

    Constrained spectral clustering with affinity propagation in its original form is not practical for large scale problems like image segmentation. In this paper we employ novelty selection sub-sampling strategy, besides using efficient numerical eigen-decomposition methods to make this algorithm work efficiently for images. In addition, entropy-based active learning is also employed to select the queries posed to the user more wisely in an interactive image segmentation framework. We evaluate the algorithm on general and medical images to show that the segmentation results will improve using constrained clustering even if one works with a subset of pixels. Furthermore, this happens more efficiently when pixels to be labeled are selected actively. PMID:24466500

  2. Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy.

    PubMed

    Masters, B R; So, P T; Gratton, E

    1998-02-09

    Two-photon excitation microscopy has the potential as an effective, noninvasive, diagnostic tool for in vivo examination of human deep tissue structure at the subcellular level. By using infrared photons as the excitation source in two-photon microscopy, a significant improvement in penetration depth can be achieved because of the much lower tissue scattering and absorption coefficients in the infrared wavelengths. Two-photon absorption occurs primarily at the focal point and provides the physical basis for optical sectioning. Multiphoton excitation microscopy at 730 nm was used to image in vivo human skin autofluorescence from the surface to a depth of about 200 microns. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence using 730 nm excitation. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells and provides a functional and morphological optical biopsy.

  3. Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

    NASA Astrophysics Data System (ADS)

    Skala, Melissa Caroline

    2007-12-01

    Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in

  4. A novel model for ectopic, chronic, intravital multiphoton imaging of bone marrow vasculature and architecture in split femurs

    PubMed Central

    Bălan, Mirela; Kiefer, Friedemann

    2015-01-01

    Creating a model for intravital visualization of femoral bone marrow, a major site of hematopoiesis in adult mammalian organisms, poses a serious challenge, in that it needs to overcome bone opacity and the inaccessibility of marrow. Furthermore, meaningful analysis of bone marrow developmental and differentiation processes requires the repetitive observation of the same site over long periods of time, which we refer to as chronic imaging. To surmount these issues, we developed a chronic intravital imaging model that allows the observation of split femurs, ectopically transplanted into a dorsal skinfold chamber of a host mouse. Repeated, long term observations are facilitated by multiphoton microscopy, an imaging technique that combines superior imaging capacity at greater tissue depth with low phototoxicity. The transplanted, ectopic femur was stabilized by its sterile environment and rapidly connected to the host vasculature, allowing further development and observation of extended processes. After optimizing transplant age and grafting procedure, we observed the development of new woven bone and maturation of secondary ossification centers in the transplanted femurs, preceded by the sprouting of a sinusoidal-like vascular network, which was almost entirely composed of femoral endothelial cells. After two weeks, the transplant was still populated with stromal and haematopoietic cells belonging both to donor and host. Over this time frame, the transplant partially retained myeloid progenitor cells with single and multi-lineage differentiation capacity. In summary, our model allowed repeated intravital imaging of bone marrow angiogenesis and hematopoiesis. It represents a promising starting point for the development of improved chronic optical imaging models for femoral bone marrow. PMID:28243515

  5. Quantifying local heterogeneity of in vivo transport dynamics using stochastic scanning multiphoton multifocal microscopy and segmented spatiotemporal image correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Kim, Hee Y.; Jureller, Justin E.; Kuznetsov, Andrey; Philipson, Louis H.; Scherer, Norbert F.

    2008-02-01

    Elucidating the mechanisms of insulin granule trafficking in pancreatic β-cells is a critical step in understanding Type II Diabetes and abnormal insulin secretion. In this paper, rapid-sampling stochastic scanning multiphoton multifocal microscopy (SS-MMM) was developed to capture fast insulin granule dynamics in vivo. Stochastic scanning of (a diffractive optic generated) 10×10 hexagonal array of foci with a galvanometer yields a uniformly sampled image with fewer spatio-temporal artifacts than obtained by conventional or multibeam raster scanning. In addition, segmented spatio-temporal image correlation spectroscopy (Segmented STICS) was developed to extract dynamics of insulin granules from the image sequences. Measurements we conducted on MIN6 cells, which exhibit an order of magnitude lower granule number density, allow comparison of particle tracking with Segmented-STICS. Segmentation of the images into 8×8 pixel segments (similar to a size of one granule) allows some amount of spatial averaging, which can reduce the computation time required to calculate the correlation function, yet retains information about the local spatial heterogeneity of transport. This allows the correlation analysis to quantify the dynamics within each of the segments producing a "map" of the localized properties of the cell. The results obtained from Segmented STICS are compared with dynamics determined from particle tracking analysis of the same images. The resulting range of diffusion coefficients of insulin granules are comparable to previously published values indicating that SS-MMM and segmented- STICS will be useful to address the imaging challenges presented by β-cells, particularly the extremely large number density of granules.

  6. Using spectral information in forensic imaging.

    PubMed

    Miskelly, Gordon M; Wagner, John H

    2005-12-20

    Improved detection of forensic evidence by combining narrow band photographic images taken at a range of wavelengths is dependent on the substance of interest having a significantly different spectrum from the underlying substrate. While some natural substances such as blood have distinctive spectral features which are readily distinguished from common colorants, this is not true for visualization agents commonly used in forensic science. We now show that it is possible to select reagents with narrow spectral features that lead to increased visibility using digital cameras and computer image enhancement programs even if their coloration is much less intense to the unaided eye than traditional reagents. The concept is illustrated by visualising latent fingermarks on paper with the zinc complex of Ruhemann's Purple, cyanoacrylate-fumed fingerprints with Eu(tta)(3)(phen), and soil prints with 2,6-bis(benzimidazol-2-yl)-4-[4'-(dimethylamino)phenyl]pyridine [BBIDMAPP]. In each case background correction is performed at one or two wavelengths bracketing the narrow absorption or emission band of these compounds. However, compounds with sharp spectral features would also lead to improved detection using more advanced algorithms such as principal component analysis.

  7. Near Infrared (nir) Spectral Imaging for Nde

    NASA Astrophysics Data System (ADS)

    Diamond, G. G.; Hutchins, D. A.; Pallav, P.; Gohel, R.

    2009-03-01

    A novel technique of NIR imaging is presented that gives access to most of the applications currently published as being solely suitable for terahertz waves. This technique also affords the means to provide simultaneous insitu chemical-bond analysis and simultaneously combine chemical/spectral identification with imaging. The two separate features of the technique can be combined in a data fusion that produces a conventional image with chemical data etc superimposed (e.g. via false colours on the image) by imaging software. The technique itself uses NIR beams wavelengths found in ordinary domestic remote controls (circa 850 nm) and various signal recovery techniques commonly found in astronomy. This alternative technique can be realised by very simple and inexpensive electronics and is inherently far more portable and easy to use and no special sources are required. Transmission imaging results from this technique are presented from several industrial examples and various security applications and are compared and contrasted directly with their terahertz-derived counterparts. It would appear possible to very cheaply and simply emulate the performance of commercial terahertz systems at a fraction of the cost and with greatly reduced processing times. Also, unlike terahertz, this technique can penetrate bulk water and high humidity atmospheres and be used in transmission mode on biological and medical samples. To illustrate this point, several results are presented of non-ionising x-ray type images that even differentiate between separate types of soft tissue.

  8. Ultrafast Imaging using Spectral Resonance Modulation

    NASA Astrophysics Data System (ADS)

    Huang, Eric; Ma, Qian; Liu, Zhaowei

    2016-04-01

    CCD cameras are ubiquitous in research labs, industry, and hospitals for a huge variety of applications, but there are many dynamic processes in nature that unfold too quickly to be captured. Although tradeoffs can be made between exposure time, sensitivity, and area of interest, ultimately the speed limit of a CCD camera is constrained by the electronic readout rate of the sensors. One potential way to improve the imaging speed is with compressive sensing (CS), a technique that allows for a reduction in the number of measurements needed to record an image. However, most CS imaging methods require spatial light modulators (SLMs), which are subject to mechanical speed limitations. Here, we demonstrate an etalon array based SLM without any moving elements that is unconstrained by either mechanical or electronic speed limitations. This novel spectral resonance modulator (SRM) shows great potential in an ultrafast compressive single pixel camera.

  9. Ultrafast Imaging using Spectral Resonance Modulation

    PubMed Central

    Huang, Eric; Ma, Qian; Liu, Zhaowei

    2016-01-01

    CCD cameras are ubiquitous in research labs, industry, and hospitals for a huge variety of applications, but there are many dynamic processes in nature that unfold too quickly to be captured. Although tradeoffs can be made between exposure time, sensitivity, and area of interest, ultimately the speed limit of a CCD camera is constrained by the electronic readout rate of the sensors. One potential way to improve the imaging speed is with compressive sensing (CS), a technique that allows for a reduction in the number of measurements needed to record an image. However, most CS imaging methods require spatial light modulators (SLMs), which are subject to mechanical speed limitations. Here, we demonstrate an etalon array based SLM without any moving elements that is unconstrained by either mechanical or electronic speed limitations. This novel spectral resonance modulator (SRM) shows great potential in an ultrafast compressive single pixel camera. PMID:27122101

  10. Ultrafast Imaging using Spectral Resonance Modulation.

    PubMed

    Huang, Eric; Ma, Qian; Liu, Zhaowei

    2016-04-28

    CCD cameras are ubiquitous in research labs, industry, and hospitals for a huge variety of applications, but there are many dynamic processes in nature that unfold too quickly to be captured. Although tradeoffs can be made between exposure time, sensitivity, and area of interest, ultimately the speed limit of a CCD camera is constrained by the electronic readout rate of the sensors. One potential way to improve the imaging speed is with compressive sensing (CS), a technique that allows for a reduction in the number of measurements needed to record an image. However, most CS imaging methods require spatial light modulators (SLMs), which are subject to mechanical speed limitations. Here, we demonstrate an etalon array based SLM without any moving elements that is unconstrained by either mechanical or electronic speed limitations. This novel spectral resonance modulator (SRM) shows great potential in an ultrafast compressive single pixel camera.

  11. Multiphoton cryo microscope with sample temperature control

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2013-02-01

    We present a multiphoton microscope system which combines the advantages of multiphoton imaging with precise control of the sample temperature. The microscope provides online insight in temperature-induced changes and effects in plant tissue and animal cells with subcellular resolution during cooling and thawing processes. Image contrast is based on multiphoton fluorescence intensity or fluorescence lifetime in the range from liquid nitrogen temperature up to +600°C. In addition, micro spectra from the imaged regions can be recorded. We present measurement results from plant leaf samples as well as Chinese hamster ovary cells.

  12. Optical Spectroscopy and Multiphoton Imaging for the Diagnosis and Characterization of Hyperplasias in the Mouse Mammary Gland

    DTIC Science & Technology

    2007-09-01

    epithelial tissues in vivo using diffuse reflectance spectroscopy . Optics Express. Accepted (2007). Thesis • MC Skala. “Multiphoton Microscopy...breast cancer using diffuse reflectance spectroscopy : Comparison of a Monte Carlo versus partial least squares analysis based feature extraction

  13. A new method using multiphoton imaging and morphometric analysis for differentiating chromophobe renal cell carcinoma and oncocytoma kidney tumors

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Mukherjee, Sushmita; Jain, Manu

    2016-03-01

    Distinguishing chromophobe renal cell carcinoma (chRCC) from oncocytoma on hematoxylin and eosin images may be difficult and require time-consuming ancillary procedures. Multiphoton microscopy (MPM), an optical imaging modality, was used to rapidly generate sub-cellular histological resolution images from formalin-fixed unstained tissue sections from chRCC and oncocytoma.Tissues were excited using 780nm wavelength and emission signals (including second harmonic generation and autofluorescence) were collected in different channels between 390 nm and 650 nm. Granular structure in the cell cytoplasm was observed in both chRCC and oncocytoma. Quantitative morphometric analysis was conducted to distinguish chRCC and oncocytoma. To perform the analysis, cytoplasm and granules in tumor cells were segmented from the images. Their area and fluorescence intensity were found in different channels. Multiple features were measured to quantify the morphological and fluorescence properties. Linear support vector machine (SVM) was used for classification. Re-substitution validation, cross validation and receiver operating characteristic (ROC) curve were implemented to evaluate the efficacy of the SVM classifier. A wrapper feature algorithm was used to select the optimal features which provided the best predictive performance in separating the two tissue types (classes). Statistical measures such as sensitivity, specificity, accuracy and area under curve (AUC) of ROC were calculated to evaluate the efficacy of the classification. Over 80% accuracy was achieved as the predictive performance. This method, if validated on a larger and more diverse sample set, may serve as an automated rapid diagnostic tool to differentiate between chRCC and oncocytoma. An advantage of such automated methods are that they are free from investigator bias and variability.

  14. Optical biopsy in high-speed handheld miniaturized multifocal multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Daekeun; Kim, Ki Hean; Yazdanfar, Siavash; So, Peter T. C.

    2005-03-01

    Histological analysis is the clinical standard for assessing tissue health and the identification of pathological states. Its invasive nature dictates that its use should be minimized without compromising diagnostic accuracy. A promising method for minimally invasive histological analysis is optical biopsy, which provides cross sectional or 3D images without any physical sectionings. Optical biopsy method based on multiphoton excitation microscopy can image cross-sectional image for deep tissue structures with subcellular resolution based on tissue endogenous fluorescence molecules. Despite its suitability for tissue imaging, multiphoton microscopy has not been used for in vivo clinical applications due to both compactness and imaging speed problems. We are developing a high-speed, handheld, miniaturized multifocal multiphoton microscope (H2M4) as an optical biopsy probe to enable optical biopsy with subcellular resolution. We incorporate a compact raster scanning actuator based on optimizing a piezo-driven tip tilt mirror by increasing its bandwidth, and reducing its nonlinearity. For flexible light delivery, we choose a photonic bandgap crystal fiber, which transmits ultrashort pulsed laser delivery with reduced spectral distortion and pulse width broadening. We further demonstrate that this fiber produces minimal spatial mode distortion and can achieve comparable image point spread function (PSF) as free space delivery. We further investigate the applicability of multiphoton microscopy for clinical dermal investigation by imaging ex vivo human skins with both normal and abnormal physiologies. This demonstrates the performance of H2M4 and the possibility of optical biopsy for diagnosing skin diseases.

  15. Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

    PubMed Central

    Peusquens, Julia; Ergen, Can; Kohlhepp, Marlene; Mossanen, Jana C.; Schneider, Carlo; Vogt, Michael; Tolba, Rene H.; Trautwein, Christian; Martin, Christian; Tacke, Frank

    2015-01-01

    Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring. The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange. To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4). CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a

  16. HYMOSS signal processing for pushbroom spectral imaging

    NASA Technical Reports Server (NTRS)

    Ludwig, David E.

    1991-01-01

    The objective of the Pushbroom Spectral Imaging Program was to develop on-focal plane electronics which compensate for detector array non-uniformities. The approach taken was to implement a simple two point calibration algorithm on focal plane which allows for offset and linear gain correction. The key on focal plane features which made this technique feasible was the use of a high quality transimpedance amplifier (TIA) and an analog-to-digital converter for each detector channel. Gain compensation is accomplished by varying the feedback capacitance of the integrate and dump TIA. Offset correction is performed by storing offsets in a special on focal plane offset register and digitally subtracting the offsets from the readout data during the multiplexing operation. A custom integrated circuit was designed, fabricated, and tested on this program which proved that nonuniformity compensated, analog-to-digital converting circuits may be used to read out infrared detectors. Irvine Sensors Corporation (ISC) successfully demonstrated the following innovative on-focal-plane functions that allow for correction of detector non-uniformities. Most of the circuit functions demonstrated on this program are finding their way onto future IC's because of their impact on reduced downstream processing, increased focal plane performance, simplified focal plane control, reduced number of dewar connections, as well as the noise immunity of a digital interface dewar. The potential commercial applications for this integrated circuit are primarily in imaging systems. These imaging systems may be used for: security monitoring systems, manufacturing process monitoring, robotics, and for spectral imaging when used in analytical instrumentation.

  17. Ultrafast pulse-pair control in multiphoton fluorescence laser-scanning microscopy.

    PubMed

    De, Arijit Kumar; Goswami, Debabrata

    2009-01-01

    In multiphoton fluorescence laser-scanning microscopy, ultrafast laser pulses [i.e., light pulses having pulse width multiphoton absorption cross-sections of common fluorophores. Because of the broad overlapping two-photon absorption spectra of fluorophores and the large spectral bandwidth of a short pulse, simultaneous excitation of many fluorophores is common, which justifies a persistent demand for selective excitation of individual fluorophores. We describe the use of pulse-pair excitation with possibilities of controlling molecular fluorescence in laser-scanning microscopy and compare it with coherent control using pulse sequence [De and Goswami, "Coherent control in multiphoton fluorescence imaging," Proc. SPIE 7183, 71832B (2009)].

  18. Detection limits with spectral differential imaging data

    NASA Astrophysics Data System (ADS)

    Rameau, J.; Chauvin, G.; Lagrange, A.-M.; Maire, A.-L.; Boccaletti, A.; Bonnefoy, M.

    2015-09-01

    Context. Direct imaging of exoplanets is polluted by speckle noise that severely limits the achievable contrast. Angular and spectral differential imaging have been proposed to make use of the temporal and chromatic properties of the speckles. Both modes, associated with extreme adaptive-optics and coronagraphy, are at the core of the new generation of planet imagers SPHERE and GPI. Aims: We aim to illustrate and characterize the impact of the SDI and SDI+ADI (ASDI) data reduction on the detection of giant planets. We also propose an unbiased method to derive the detection limits from SDI/ASDI data. Methods: Observations of AB Dor B and β Pictoris made with VLT/NaCo were used to simulate and quantify the effects of SDI and ASDI. The novel method is compared to the traditional injection of artificial point sources. Results: The SDI reduction process creates a typical radial positive-negative pattern of any point-source. Its characteristics and its self-subtraction depend on the separation, but also on the spectral properties of the object. This work demonstrates that the self-subtraction cannot be reduced to a simple geometric effect. As a consequence, the detection performances of SDI observations cannot be expressed as a contrast in magnitude with the central star without the knowledge of the spectral properties of detectable companions. In addition, the residual noise cannot be converted into contrast and physical characteristics (mass, temperature) by standard calibration of flux losses. The proposed method takes the SDI bias into account to derive detection limits without the cost of massively injecting artificial sources into the data. Finally, the sensitivity of ASDI observations can be measured only with a control parameter on the algorithms that controls the minimum rotation that is necessary to build the reference image. Based on observations collected at the European Organization for Astronomical Research in the Southern Hemisphere, Chile, ESO : 60.A

  19. Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

    DTIC Science & Technology

    2008-03-01

    observations, quantitative analysis of whole mounts of the fourth mammary gland (n = 3 pairs) show significantly increased areas of hyper - plasia (Figure...ated signaling pathways are often interrelated [52-57], understanding each of these possible mechanisms and their convergence is likely to be of...interest, can be cleanly obtained Fig. 5. However, in order to efficiently analyze the large multidimensional data sets from SLIM, a computational infra

  20. Spectral Camera based on Ghost Imaging via Sparsity Constraints

    PubMed Central

    Liu, Zhentao; Tan, Shiyu; Wu, Jianrong; Li, Enrong; Shen, Xia; Han, Shensheng

    2016-01-01

    The image information acquisition ability of a conventional camera is usually much lower than the Shannon Limit since it does not make use of the correlation between pixels of image data. Applying a random phase modulator to code the spectral images and combining with compressive sensing (CS) theory, a spectral camera based on true thermal light ghost imaging via sparsity constraints (GISC spectral camera) is proposed and demonstrated experimentally. GISC spectral camera can acquire the information at a rate significantly below the Nyquist rate, and the resolution of the cells in the three-dimensional (3D) spectral images data-cube can be achieved with a two-dimensional (2D) detector in a single exposure. For the first time, GISC spectral camera opens the way of approaching the Shannon Limit determined by Information Theory in optical imaging instruments. PMID:27180619

  1. Evaluating collagen morphology and pathological lipid deposition using multiphoton image statistics

    NASA Astrophysics Data System (ADS)

    Mostaço-Guidolin, Leila B.; Ko, Alex C.-T.; Wang, Fei; Tian, Hong; Hewko, Mark; Shiomi, Masashi; Major, Arkady; Sowa, Michael G.

    2014-02-01

    In this study we present a novel image analysis methodology to quantify and to classify morphological details in tissue collagen fibril organization and lipid deposition. Co-localized collagen (second harmonic, SHG) and lipid (coherent Raman, CARS) images of atherosclerotic artery walls were acquired by a supercontinuum-powered multi-modal nonlinear microscope. Textural features based on the first-order statistics (FOS) and gray level co-occurrence matrix (GLCM) parameters were extracted from the SHG and CARS images. Multi-group classifications based on support vector machine of SHG and CARS images were subsequently performed to investigate the potential of texture analysis in providing quantitative descriptors of structural and compositional changes during disease progression. Using a rabbit model, different collagen remodeling and lipid accumulation patterns in disease tissues can be successfully tracked using these image statistics, thus providing a robust foundation for classification. When the variation of the CARS image features were tracked against the age of the rabbit, it was noticed that older animals (advanced plaques) present a more complex necrotic core containing high-lipid extracellular structures with various shapes and distribution. With combined FOS and GLCM texture statistics, we achieved reliable classification of SHG and CARS images acquired from atherosclerotic arteries with >90% accuracy, sensitivity and specificity. The proposed image analysis methodology can also be applied in a wide range of applications to evaluate conditions involving collagen re-modeling and prominent lipid accumulation.

  2. Mosaic-Detector-Based Fluorescence Spectral Imager

    NASA Technical Reports Server (NTRS)

    Son, Kyung-Ah; Moon, Jeong

    2007-01-01

    A battery-powered, pen-sized, portable instrument for measuring molecular fluorescence spectra of chemical and biological samples in the field has been proposed. Molecular fluorescence spectroscopy is among the techniques used most frequently in laboratories to analyze compositions of chemical and biological samples. Heretofore, it has been possible to measure fluorescence spectra of molecular species at relative concentrations as low as parts per billion (ppb), with a few nm spectral resolution. The proposed instrument would include a planar array (mosaic) of detectors, onto which a fluorescence spectrum would be spatially mapped. Unlike in the larger laboratory-type molecular fluorescence spectrometers, mapping of wavelengths to spatial positions would be accomplished without use of relatively bulky optical parts. The proposed instrument is expected to be sensitive enough to enable measurement of spectra of chemical species at relative concentrations <1 ppb, with spectral resolution that could be tailored by design to be comparable to a laboratory molecular fluorescence spectrometer. The proposed instrument (see figure) would include a button-cell battery and a laser diode, which would generate the monochromatic ultraviolet light needed to excite fluorescence in a sample. The sample would be held in a cell bounded by far-ultraviolet-transparent quartz or optical glass. The detector array would be, more specifically, a complementary metal oxide/ semiconductor or charge-coupled- device imaging photodetector array, the photodetectors of which would be tailored to respond to light in the wavelength range of the fluorescence spectrum to be measured. The light-input face of the photodetector array would be covered with a matching checkerboard array of multilayer thin film interference filters, such that each pixel in the array would be sensitive only to light in a spectral band narrow enough so as not to overlap significantly with the band of an adjacent pixel. The

  3. An upper-bound metric for characterizing spectral and spatial coregistration errors in spectral imaging.

    PubMed

    Skauli, Torbjørn

    2012-01-16

    Coregistration errors in multi- and hyperspectral imaging sensors arise when the spatial sensitivity pattern differs between bands or when the spectral response varies across the field of view, potentially leading to large errors in the recorded image data. In imaging spectrometers, spectral and spatial offset errors are customarily specified as "smile" and "keystone" distortions. However these characteristics do not account for errors resulting from variations in point spread function shape or spectral bandwidth. This paper proposes improved metrics for coregistration error both in the spatial and spectral dimensions. The metrics are essentially the integrated difference between point spread functions. It is shown that these metrics correspond to an upper bound on the error in image data. The metrics enable estimation of actual data errors for a given image, and can be used as part of the merit function in optical design optimization, as well as for benchmarking of spectral image sensors.

  4. Motion quantification during multi-photon functional imaging in behaving animals

    PubMed Central

    Kong, Lingjie; Little, Justin P.; Cui, Meng

    2016-01-01

    Functional imaging in behaving animals is essential to understanding brain function. However, artifacts resulting from animal motion, including locomotion, can severely corrupt functional measurements. To dampen tissue motion, we designed a new optical window with minimal optical aberrations. Using the newly developed high-speed continuous volumetric imaging system based on an optical phase-locked ultrasound lens, we quantified motion of the cerebral cortex and hippocampal surface during two-photon functional imaging in behaving mice. We find that the out-of-plane motion is generally greater than the axial dimension of the point-spread-function during mouse locomotion, which indicates that high-speed continuous volumetric imaging is necessary to minimize motion artifacts. PMID:27699129

  5. High spectral resolution image of Barnacle Bill

    NASA Technical Reports Server (NTRS)

    1997-01-01

    The rover Sojourner's first target for measurement by the Alpha-Proton-Xray Spectrometer (APXS) was the rock named Barnacle Bill, located close to the ramp down which the rover made its egress from the lander. The full spectral capability of the Imager for Mars Pathfinder (IMP), consisting of 13 wavelength filters, was used to characterize the rock's surface. The measured area is relatively dark, and is shown in blue. Nearby on the rock surface, soil material is trapped in pits (shown in red).

    Mars Pathfinder is the second in NASA's Discovery program of low-cost spacecraft with highly focused science goals. The Jet Propulsion Laboratory, Pasadena, CA, developed and manages the Mars Pathfinder mission for NASA's Office of Space Science, Washington, D.C. The Imager for Mars Pathfinder (IMP) was developed by the University of Arizona Lunar and Planetary Laboratory under contract to JPL. Peter Smith is the Principal Investigator. JPL is an operating division of the California Institute of Technology (Caltech).

  6. Monte Carlo simulation of multiphoton fluorescence microscopic imaging through inhomogeneous tissuelike turbid media.

    PubMed

    Deng, Xiaoyuan; Gan, Xiaosong; Gu, Min

    2003-07-01

    Image resolution and signal level in fluorescence microscopy through inhomogeneous turbid media consisting of scatterers of multiple sizes under single- (1p), two- (2p), and three-photon (3p) excitation have been investigated based on a modified Monte Carlo model. The effects of the size distribution and the concentration distribution of scattering particles are explored. Simulation results reveal that the size and the concentration distribution both have an impact on image formation in media consisting of small particles and that 3p excitation has the most significant impact. In media with scatterers of a large size, both size and concentration distributions lead to a slight effect. Image formation in a mixed medium containing small and large scattering particles is more affected by the large particles.

  7. A novel clinical multimodal multiphoton tomograph for AF, SHG, CARS imaging, and FLIM

    NASA Astrophysics Data System (ADS)

    Weinigel, Martin; Breunig, Hans Georg; König, Karsten

    2014-02-01

    We report on a flexible nonlinear medical tomograph with multiple miniaturized detectors for simultaneous acquisition of two-photon autofluorescence (AF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) images. The simultaneous visualization of the distribution of endogenous fluorophores NAD(P)H, melanin and elastin, SHG-active collagen and as well as non-fluorescent lipids within human skin in vivo is possible. Furthermore, fluorescence lifetime images (FLIM) can be generated using time-correlated single photon counting.

  8. Spectral imaging of breast fibroadenoma using second-harmonic generation

    NASA Astrophysics Data System (ADS)

    Zheng, Liqin; Wang, Yuhua

    2014-09-01

    Fibroadenoma (FA), typically composed of stroma and epithelial cells, is a very common benign breast disease. Women with FA are associated with an increased risk of future breast cancer. The objective of this study was to demonstrate the potential of multiphoton laser scanning microscopy (MPLSM) for characterizing the morphology of collagen in the human breast fibroadenomas. In the study, high-contrast SHG images of human normal breast tissues and fibroadenoma tissues were obtained for comparison. The morphology of collagen was different between normal breast tissue and fibroadenoma. This study shows that MPLSM has the ability to distinguish fibroadenoma tissues from the normal breast tissues based on the noninvasive SHG imaging. With the advent of the clinical portability of miniature MPLSM, we believe that the technique has great potential to be used in vivo studies and for monitoring the treatment responses of fibroadenomas in clinical.

  9. Compressive spectral integral imaging using a microlens array

    NASA Astrophysics Data System (ADS)

    Feng, Weiyi; Rueda, Hoover; Fu, Chen; Qian, Chen; Arce, Gonzalo R.

    2016-05-01

    In this paper, a compressive spectral integral imaging system using a microlens array (MLA) is proposed. This system can sense the 4D spectro-volumetric information into a compressive 2D measurement image on the detector plane. In the reconstruction process, the 3D spatial information at different depths and the spectral responses of each spatial volume pixel can be obtained simultaneously. In the simulation, sensing of the 3D objects is carried out by optically recording elemental images (EIs) using a scanned pinhole camera. With the elemental images, a spectral data cube with different perspectives and depth information can be reconstructed using the TwIST algorithm in the multi-shot compressive spectral imaging framework. Then, the 3D spatial images with one dimensional spectral information at arbitrary depths are computed using the computational integral imaging method by inversely mapping the elemental images according to geometrical optics. The simulation results verify the feasibility of the proposed system. The 3D volume images and the spectral information of the volume pixels can be successfully reconstructed at the location of the 3D objects. The proposed system can capture both 3D volumetric images and spectral information in a video rate, which is valuable in biomedical imaging and chemical analysis.

  10. USE OF MULTIPHOTON LASER SCANNING MICROSCOPY TO IMAGE BENZO[A]PYRENE AND METABOLITES IN FISH EARLY LIFE STAGES

    EPA Science Inventory

    Multiphoton laser scanning micrsocopy holds promise as a tool to study the tissue distribution of environmental chemical contaminants during fish early life stage development. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polyaromatic hydrocarbon that a...

  11. USE OF MULTIPHOTON LASER SCANNING MICROSCOPY TO IMAGE BENZO[A]PYRENE AND METABOLITES IN FISH EGGS

    EPA Science Inventory

    Multiphoton laser scanning microscopy (MPLSM) is a promising tool to study the tissue distribution of environmental chemical contaminants during fish early life stages. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon that a...

  12. Bleed-through and photobleaching correction in multiphoton FRET microscopy

    NASA Astrophysics Data System (ADS)

    Elangovan, Masilamani; Periasamy, Ammasi

    2001-04-01

    Fluorescence resonance energy transfer (FRET) microscopy provides a tool to visualize the protein with high spatial and temporal resolution. In multi-photon FRET microscopy one experiences considerably less photobleaching compared to one-photon excitation since the illumination is the diffraction limited spot and the excitation is infrared-pulsed laser light. Because of the spectral overlap involved in the selection of the fluorophore pair for FRET imaging, the spectral bleed-through signal in the FRET channel is unavoidable. We describe in this paper the development of dedicated software to correct the bleed-through signal due to donor and acceptor fluorophore molecules. We used living cells expressed with BFP-RFP (DsRed)-C/EBP(alpha) proteins in the nucleus. We acquired images of different combinations like donor alone, acceptor alone, and both acceptor and donor under similar conditions. We statistically evaluated the percentage of bleed-through signal from one channel to the other based on the overlap areas of the spectra. We then reconstructed the images after applying the correction. Characterization of multi-photon FRET imaging system taking into account the intensity, dwell time, concentration of fluorophore pairs, objective lens and the excitation wavelength are described in this paper.

  13. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

    NASA Astrophysics Data System (ADS)

    Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

    2016-03-01

    Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

  14. A New Multichannel Spectral Imaging Laser Scanning Confocal Microscope

    PubMed Central

    Zhang, Yunhai; Hu, Bian; Dai, Yakang; Yang, Haomin; Huang, Wei; Xue, Xiaojun; Li, Fazhi; Zhang, Xin; Jiang, Chenyu; Gao, Fei; Chang, Jian

    2013-01-01

    We have developed a new multichannel spectral imaging laser scanning confocal microscope for effective detection of multiple fluorescent labeling in the research of biological tissues. In this paper, the design and key technologies of the system are introduced. Representative results on confocal imaging, 3-dimensional sectioning imaging, and spectral imaging are demonstrated. The results indicated that the system is applicable to multiple fluorescent labeling in biological experiments. PMID:23585775

  15. Hyperspectral retinal imaging with a spectrally tunable light source

    NASA Astrophysics Data System (ADS)

    Francis, Robert P.; Zuzak, Karel J.; Ufret-Vincenty, Rafael

    2011-03-01

    Hyperspectral retinal imaging can measure oxygenation and identify areas of ischemia in human patients, but the devices used by current researchers are inflexible in spatial and spectral resolution. We have developed a flexible research prototype consisting of a DLP®-based spectrally tunable light source coupled to a fundus camera to quickly explore the effects of spatial resolution, spectral resolution, and spectral range on hyperspectral imaging of the retina. The goal of this prototype is to (1) identify spectral and spatial regions of interest for early diagnosis of diseases such as glaucoma, age-related macular degeneration (AMD), and diabetic retinopathy (DR); and (2) define required specifications for commercial products. In this paper, we describe the challenges and advantages of using a spectrally tunable light source for hyperspectral retinal imaging, present clinical results of initial imaging sessions, and describe how this research can be leveraged into specifying a commercial product.

  16. Complex spectral OCT in human eye imaging in vivo

    NASA Astrophysics Data System (ADS)

    Targowski, Piotr; Wojtkowski, Maciej; Kowalczyk, Andrzej; Bajraszewski, Tomasz; Szkulmowski, Maciej; Gorczyńska, Iwona

    2004-01-01

    Complex spectral optical coherence tomography in comparison to spectral optical coherence tomography produces images free of parasitic terms with extended measurement range. This technique requires stability of the object during at least three consecutive measurements. In this paper we present how to improve this technique to make measurements less sensitive to involuntary eye movements. The first images of human skin and anterior chamber of the eye in vivo based on complex spectral optical coherence tomography are presented.

  17. Multiphoton microscopy in life sciences.

    PubMed

    König, K

    2000-11-01

    Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm(-2) to GW cm(-2), which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth-resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a one-photon or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via

  18. Passive Microwave Spectral Imaging of Amospheric Structure

    NASA Technical Reports Server (NTRS)

    Staelin, David H.; Rosenkranz, Philip W.

    1998-01-01

    The primary objective of this research was to improve the scientific foundation necessary to full realization of the meteorological potential of the NOAA Advanced Microwave Sounding Unit (AMSU) recently first launched on the NOAA-15 satellite in May, 1998. These advances were made in four main areas: (1) improvements, based on aircraft observations, in the atmospheric transmittance expressions used for interpreting AMSU and similar data; (2) development of neural network retrieval methods for cloud top altitude estimates of approximately 1-km accuracy under cirrus shields--the altitude is that of the larger ice particles aloft, which is related to precipitation rate; (3) analysis of early AMSU flight data with respect to its precipitation sensitivity and fine-scale thermal structure; and (4) improvements to the 54-GHz and 118-GHz MTS aircraft imaging spectrometer now operating on the NASA ER-2 aircraft. More specifically, the oxygen transmittance expressions near 118 GHz were in better agreement with aircraft data when the temperature dependence exponent of the 118.75-GHz linewidth was increased from the MPM92 value (Liebe et al., 1992) of 0.8 to 0.97+/-0.03. In contrast, the observations 52.5-55.8 GHz were consistent with the MPM92 model. Neural networks trained on comparisons of 118-GHz spectral data and coincident stereoscopic video images of convective cells observed from 20-km altitude yielded agreement in their peak altitudes within as little as 1.36 km rms, much of which is stereoscopic error. Imagery using these methods produced useful characterizations for Cyclone Oliver in 1993 and other storms (Schwartz et al., 1996; Spina et al., 1998). Similar neural network techniques yielded simulated rms errors in relative humidity retrievals of 6-14 percent over ocean and 6-15 percent over land at pressure levels from 1013 to 131 mbar (Cabrera-Mercader and Staelin, 1995).

  19. A custom image-based analysis tool for quantifying elastin and collagen micro-architecture in the wall of the human aorta from multi-photon microscopy.

    PubMed

    Koch, Ryan G; Tsamis, Alkiviadis; D'Amore, Antonio; Wagner, William R; Watkins, Simon C; Gleason, Thomas G; Vorp, David A

    2014-03-21

    The aorta possesses a micro-architecture that imparts and supports a high degree of compliance and mechanical strength. Alteration of the quantity and/or arrangement of the main load-bearing components of this micro-architecture--the elastin and collagen fibers--leads to mechanical, and hence functional, changes associated with aortic disease and aging. Therefore, in the future, the ability to rigorously characterize the wall fiber micro-architecture could provide insight into the complicated mechanisms of aortic wall remodeling in aging and disease. Elastin and collagen fibers can be observed using state-of-the-art multi-photon microscopy. Image-analysis algorithms have been effective at characterizing fibrous constructs using various microscopy modalities. The objective of this study was to develop a custom MATLAB-language automated image-based analysis tool to describe multiple parameters of elastin and collagen micro-architecture in human soft fibrous tissue samples using multi-photon microscopy images. Human aortic tissue samples were used to develop the code. The tool smooths, cleans and equalizes fiber intensities in the image before segmenting the fibers into a binary image. The binary image is cleaned and thinned to a fiber skeleton representation of the image. The developed software analyzes the fiber skeleton to obtain intersections, fiber orientation, concentration, porosity, diameter distribution, segment length and tortuosity. In the future, the developed custom image-based analysis tool can be used to describe the micro-architecture of aortic wall samples in a variety of conditions. While this work targeted the aorta, the software has the potential to describe the architecture of other fibrous materials, tube-like networks and connective tissues.

  20. Multiscale structural analysis of mouse lingual myoarchitecture employing diffusion spectrum magnetic resonance imaging and multiphoton microscopy.

    PubMed

    Gaige, Terry A; Kwon, Hyuk Sang; Dai, Guangping; Cabral, Victor C; Wang, Ruopeng; Nam, Yoon Sung; Engelward, Bevin P; Wedeen, Van J; So, Peter T C; Gilbert, Richard J

    2008-01-01

    The tongue consists of a complex, multiscale array of myofibers that comprise the anatomical underpinning of lingual mechanical function. 3-D myoarchitecture was imaged in mouse tongues with diffusion spectrum magnetic resonance imaging (DSI) at 9.4 T (b(max) 7000 smm, 150-microm isotropic voxels), a method that derives the preferential diffusion of water/voxel, and high-throughput (10 fps) two-photon microscope (TPM). Net fiber alignment was represented for each method in terms of the local maxima of an orientational distribution function (ODF) derived from the local diffusion (DSI) and 3-D structural autocorrelation (TPM), respectively. Mesoscale myofiber tracts were generated by alignment of the principal orientation vectors of the ODFs. These data revealed a consistent relationship between the properties of the respective ODFs and the virtual superimposition of the distributed mesoscale myofiber tracts. The identification of a mesoscale anatomical construct, which specifically links the microscopic and macroscopic spatial scales, provides a method for relating the orientation and distribution of cells and subcellular components with overall tissue morphology, thus contributing to the development of multiscale methods for mechanical analysis.

  1. SPECTRAL IMAGING OF GALAXY CLUSTERS WITH PLANCK

    SciTech Connect

    Bourdin, H.; Mazzotta, P.; Rasia, E.

    2015-12-20

    The Sunyaev–Zeldovich (SZ) effect is a promising tool for detecting the presence of hot gas out to the galaxy cluster peripheries. We developed a spectral imaging algorithm dedicated to the SZ observations of nearby galaxy clusters with Planck, with the aim of revealing gas density anisotropies related to the filamentary accretion of materials, or pressure discontinuities induced by the propagation of shock fronts. To optimize an unavoidable trade-off between angular resolution and precision of the SZ flux measurements, the algorithm performs a multi-scale analysis of the SZ maps as well as of other extended components, such as the cosmic microwave background (CMB) anisotropies and the Galactic thermal dust. The demixing of the SZ signal is tackled through kernel-weighted likelihood maximizations. The CMB anisotropies are further analyzed through a wavelet analysis, while the Galactic foregrounds and SZ maps are analyzed via a curvelet analysis that best preserves their anisotropic details. The algorithm performance has been tested against mock observations of galaxy clusters obtained by simulating the Planck High Frequency Instrument and by pointing at a few characteristic positions in the sky. These tests suggest that Planck should easily allow us to detect filaments in the cluster peripheries and detect large-scale shocks in colliding galaxy clusters that feature favorable geometry.

  2. Fluorescence spectral imaging of organelle interaction

    NASA Astrophysics Data System (ADS)

    Kohen, Elli; Hirschberg, Joseph G.; Kohen, Cahide; Schachtschabel, Dietrich O.; Monti, Marco; Stanikunaite, Rita

    2000-04-01

    In cell biology, one of the great mysteries, which has bene only superficially 8investigate,d is the integration of cytoplasmic and nuclear organelles as part of the intracellular regulatory mechanism. The methodology used for the exploration of such intracellular processes is the pixel-by-pixel scanning by means of fluorescence spectral imaging and excitation emission fluorescence spectroscopy. While several of the steps required by this method are still in the process of implementation, the Michelson interferometer, the Sagnac interferometer and the related 'pentaferometer' are possible components of the instrumental design. One of the illustrative experimental models to begin the study of intracellular integrative processes is based on the hypothesis of a 'nuclear pump' in conjunction with cell treatment by chemotherapeutic agents such as adriamycin. Preliminary observations initiated in cultured fibroblasts, and to be pursued in Cloudman's melanoma cells, suggest that this cytotoxic agent first moves into the nucleus, form which it is subsequently ejected to be incorporated into the lysosomes and Golgi apparatus, possibly prior to exclusion via the multiple drug resistance pathway. The timetable of such a process is under investigation. This subject has obvious implications for diagnostic, prognostic and therapeutic studies of organelles integration.

  3. Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells

    NASA Astrophysics Data System (ADS)

    Rück, Angelika; Hauser, Carmen; Mosch, Simone; Kalinina, Sviatlana

    2014-09-01

    Fluorescence-guided diagnosis of tumor tissue is in many cases insufficient, because false positive results interfere with the outcome. Improvement through observation of cell metabolism might offer the solution, but needs a detailed understanding of the origin of autofluorescence. With respect to this, spectrally resolved multiphoton fluorescence lifetime imaging was investigated to analyze cell metabolism in metabolic phenotypes of malignant and nonmalignant oral mucosa cells. The time-resolved fluorescence characteristics of NADH were measured in cells of different origins. The fluorescence lifetime of bound and free NADH was calculated from biexponential fitting of the fluorescence intensity decay within different spectral regions. The mean lifetime was increased from nonmalignant oral mucosa cells to different squamous carcinoma cells, where the most aggressive cells showed the longest lifetime. In correlation with reports in the literature, the total amount of NADH seemed to be less for the carcinoma cells and the ratio of free/bound NADH was decreased from nonmalignant to squamous carcinoma cells. Moreover for squamous carcinoma cells a high concentration of bound NADH was found in cytoplasmic organelles (mainly mitochondria). This all together indicates that oxidative phosphorylation and a high redox potential play an important role in the energy metabolism of these cells.

  4. Digital spectral separation methods and systems for bioluminescence imaging.

    PubMed

    Wang, Ge; Shen, Haiou; Liu, Ying; Cong, Alex; Cong, Wenxiang; Wang, Yue; Dubey, Purnima

    2008-02-04

    We propose a digital spectral separation (DSS) system and methods to extract spectral information optimally from a weak multi-spectral signal such as in the bioluminescent imaging (BLI) studies. This system utilizes our newly invented spatially-translated spectral-image mixer (SSM), which consists of dichroic beam splitters, a mirror, and a DSS algorithm. The DSS approach overcomes the shortcomings of the data acquisition scheme used for the current BLI systems. Primarily, using our DSS scheme, spectral information will not be filtered out. Accordingly, truly parallel multi-spectral multi-view acquisition is enabled for the first time to minimize experimental time and optimize data quality. This approach also permits recovery of the bioluminescent signal time course, which is useful to study the kinetics of multiple bioluminescent probes using multi-spectral bioluminescence tomography (MSBT).

  5. Dynamic multiphoton imaging of reversible and irreversible thermal changes in collagen tissues

    NASA Astrophysics Data System (ADS)

    Hovhannisyan, Vladimir A.; Su, Ping-Jung; Dong, Chen-Yuan

    2011-07-01

    Collagen is the major component of skin, tendon, cartilage, cornea, and, as a main structural protein it is the key determinant of thermo-mechanical properties of collagen-rich tissues in mammals. Thermal damage of chicken dermis and tendon, bovine leg tendon, and other collagen contained tissues were investigated with the use of second harmonic generation (SHG) and two-photon excited auto-fluorescence microscopy and spectroscopy. Samples were heating in a temperature-controlled water bath in the temperature range 18-90° C. SHG time-lapse imaging and analysis of intensity decay showed that the collagen thermal destruction depended on both temperature and heating time, and can be modeled by the Arrhenius equation. Temporal decay of SHG signal from the chicken dermis was single exponential during isothermal treatment at temperatures above 60º C that allowed to determine activation energy and frequency factor of skin collagen denaturation. Furthermore, two-exponential decay and partially reversible change in SHG intensity were registered during the tendon thermal treatment. A simple laser system and procedure is proposed for a real-time monitoring of collagen fiber thermal modification within a microscopic volume of 1 nl.

  6. Elucidation of the mechanisms of optical clearing in collagen tissue with multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Hovhannisyan, Vladimir; Hu, Po-Sheng; Chen, Shean-Jen; Kim, Chang-Seok; Dong, Chen-Yuan

    2013-04-01

    Optical clearing (OC) is a promising method to overcome limitations in biomedical depth-resolved optical studies. Mechanisms of OC in purified bovine Achilles tendon, chicken skin, and chicken tendon were studied using time-lapsed, three-dimensional second harmonic generation (SHG) and two-photon fluorescence microscopic imaging. Quantified nonlinear optical measurements allowed temporal separation of two processes in collagen OC with glycerol. The first one is a fast process of tissue dehydration accompanied with collagen shrinkage and the second relatively slow process is glycerol penetration into the interfibrillar space of collagen alongside with CF swelling. The use of 50% glycerol induced less-expressed OC via partial substitution of water molecules with glycerol molecules. We also found that phosphate-buffered saline- and glycerol-treatments were reversible, and fiber morphology and SHG signal intensity were recovered after the removal of immersion agents. It was shown that tissue OC was a dynamic process and elucidation of its physical mechanisms may help choose optimal diagnostic, treatment, and modification regimes for collagen-based as well as other types of biomaterials.

  7. Hyperspectral stimulated Raman scattering and multiphoton imaging for digital pathology of colonic disease

    NASA Astrophysics Data System (ADS)

    Wang, Zi; Zheng, Wei; Lin, Jian; Huang, Zhiwei

    2016-03-01

    Histopathology examinations of H&E stained biopsied tissues is the golden standard for colonic diseases (e.g., polyps, adenoma, and adenocarcinoma) diagnosis. However, staining effect of sample and doctor's expertise degree may greatly influence the diagnosis results. The information provided by the H&E stained sample is also limited to the morphological and PH information and no quantative information is available. In this paper, we report the development of a unique multimodal nonlinear optical microscopy (i.e., hyperspectral stimulated Raman scattering (hsSRS), second-harmonic generation (SHG), third-harmonic generation (THG), two-photon excitation fluorescence (TPEF)) platform for the diagnosis and characterization of colonic diseases. HsSRS in both fingerprint (800-1800 cm-1) and high-wavenumber (2800-3600 cm-1) regions allows us to discriminate different constituents with tiny difference in the Raman spectra. The increase of proteins and reduction of lipids could be observed with the progress of colonic cancer. SHG shows the distribution of collagen, which is found to aggregate for adenocarcinoma. TPEF provides the cell morphological and can reflect the damage inside glands caused by the diseases. THG shows the increase of optical heterogeneity related to cancer process. This work shows that the integrated hsSRS and TPEF/SHG/THG imaging technique can be an effective method for digital pathology of colonic diseases at the molecular and sub-cellular levels.

  8. Elucidation of the mechanisms of optical clearing in collagen tissue with multiphoton imaging.

    PubMed

    Hovhannisyan, Vladimir; Hu, Po-Sheng; Chen, Shean-Jen; Kim, Chang-Seok; Dong, Chen-Yuan

    2013-04-01

    Optical clearing (OC) is a promising method to overcome limitations in biomedical depth-resolved optical studies. Mechanisms of OC in purified bovine Achilles tendon, chicken skin, and chicken tendon were studied using time-lapsed, three-dimensional second harmonic generation (SHG) and two-photon fluorescence microscopic imaging. Quantified nonlinear optical measurements allowed temporal separation of two processes in collagen OC with glycerol. The first one is a fast process of tissue dehydration accompanied with collagen shrinkage and the second relatively slow process is glycerol penetration into the interfibrillar space of collagen alongside with CF swelling. The use of 50% glycerol induced less-expressed OC via partial substitution of water molecules with glycerol molecules. We also found that phosphate-buffered saline- and glycerol-treatments were reversible, and fiber morphology and SHG signal intensity were recovered after the removal of immersion agents. It was shown that tissue OC was a dynamic process and elucidation of its physical mechanisms may help choose optimal diagnostic, treatment, and modification regimes for collagen-based as well as other types of biomaterials.

  9. Analysis of the dispersion compensation of acousto-optic deflectors used for multiphoton imaging.

    PubMed

    Zeng, Shaoqun; Lv, Xiaohua; Bi, Kun; Zhan, Cheng; Li, Derong; Chen, Wei R; Xiong, Wenhui; Jacques, Steven L; Luo, Qingming

    2007-01-01

    The acousto-optic deflector (AOD) is highly preferred in laser scanning microscopy for its fast scanning ability and random-addressing capability. However, its application in two-photon microscopy is frustrated by the dispersion of the AOD, which results in beam distortion and pulse lengthening. We report the analysis of simultaneous compensation for the angular dispersion and temporal dispersion of the AOD by merely introducing a single dispersive element such as a prism or a grating. Besides serving as a scanner, the AOD is also a part of the compressor pair by integrating the dispersive nature of the AO interaction. This compensation principle is effective for both one-dimensional (1-D) AOD and two-dimensional (2-D) AOD scanning. Switching from a 1-D to a 2-D system requires proper optical alignment with the compensation element, but does not involve any new components. Analytical expressions are given to illustrate the working principle and to help with understanding the design of the system. Fluorescence images of beads and cells are shown to demonstrate the performance of two-photon microscopy when applying this compensated 2-D AOD as scanner.

  10. Multiphoton microscopy of atheroslcerotic plaques

    NASA Astrophysics Data System (ADS)

    Lilledahl, Magnus B.; de Lange Davies, Catharina; Haugen, Olav A.; Svaasand, Lars O.

    2007-02-01

    Multiphoton microscopy is a techniques that fascilitates three dimensional imaging of intact, unstained tissue. Especially connective tissue has a relatively strong nonlinear optical response and can easily be imaged. Atherosclerosis is a disease where lipids accumulate in the vessel wall and there is a thickening of the intima by growth of a cap of connective tissue. The mechanical strength of this fibrous cap is of clinically importance. If the cap ruptures a thrombosis forms which can block a coronary vessel and therby causing myocardial infarction. Multiphoton microscopy can be used to image the fibrous cap and thereby determine the thickness of the cap and the structure of the connective fibres. This could possibly be developed into a diagnostic and clincal tool to monitor the vulnerability of a plaque and also to better understand the development of a plaque and effects of treatment. We have collected multiphoton microscopy images from atherosclerotic plaque in human aorta, both two photon excited fluorescens and second harmonic generated signal. The feasability of using this technique to determine the state of the plaque is explored.

  11. Fast spectral color image segmentation based on filtering and clustering

    NASA Astrophysics Data System (ADS)

    Xing, Min; Li, Hongyu; Jia, Jinyuan; Parkkinen, Jussi

    2009-10-01

    This paper proposes a fast approach to spectral image segmentation. In the algorithm, two popular techniques are extended and applied to spectral color images: the mean-shift filtering and the kernel-based clustering. We claim that segmentation should be completed under illuminant F11 rather than directly using the original spectral reflectance, because such illumination can reduce data variability and expedite the following filtering. The modes obtained in the mean-shift filtering represent the local features of spectral images, and will be applied to segmentation in place of pixels. Since the modes are generally small in number, the eigendecomposition of kernel matrices, the crucial step in the kernelbased clustering, becomes much easier. The combination of these two techniques can efficiently enhance the performance of segmentation. Experiments show that the proposed segmentation method is feasible and very promising for spectral color images.

  12. SAR image change detection using watershed and spectral clustering

    NASA Astrophysics Data System (ADS)

    Niu, Ruican; Jiao, L. C.; Wang, Guiting; Feng, Jie

    2011-12-01

    A new method of change detection in SAR images based on spectral clustering is presented in this paper. Spectral clustering is employed to extract change information from a pair images acquired on the same geographical area at different time. Watershed transform is applied to initially segment the big image into non-overlapped local regions, leading to reduce the complexity. Experiments results and system analysis confirm the effectiveness of the proposed algorithm.

  13. Multiphoton processes: conference proceedings

    SciTech Connect

    Lambropoulos, P.; Smith, S.J.

    1984-01-01

    The chapters of this volume represent the invited papers delivered at the conference. They are arranged according to thermatic proximity beginning with atoms and continuing with molecules and surfaces. Section headings include multiphoton processes in atoms, field fluctuations and collisions in multiphoton process, and multiphoton processes in molecules and surfaces. Abstracts of individual items from the conference were prepared separately for the data base. (GHT)

  14. Multiphoton microscopy: an introduction to gastroenterologists.

    PubMed

    Cho, Hye Jin; Chun, Hoon Jai; Kim, Eun Sun; Cho, Bong Rae

    2011-10-28

    Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observation of a single cell and molecules in intact tissues. Multiphoton microscopy exhibits nonlinear optical contrast properties, which can make it possible to provide an exceptionally large depth penetration with less phototoxicity. This system becomes more and more an inspiring tool for a non-invasive imaging system to realize "optical biopsy" and to examine the functions of living cells. In this review, we briefly present the physical principles and properties of multiphoton microscopy as well as the current applications in biological fields. In addition, we address what we see as the future potential of multiphoton microscopy for gastroenterologic research.

  15. Semiconductor laser multi-spectral sensing and imaging.

    PubMed

    Le, Han Q; Wang, Yang

    2010-01-01

    Multi-spectral laser imaging is a technique that can offer a combination of the laser capability of accurate spectral sensing with the desirable features of passive multispectral imaging. The technique can be used for detection, discrimination, and identification of objects by their spectral signature. This article describes and reviews the development and evaluation of semiconductor multi-spectral laser imaging systems. Although the method is certainly not specific to any laser technology, the use of semiconductor lasers is significant with respect to practicality and affordability. More relevantly, semiconductor lasers have their own characteristics; they offer excellent wavelength diversity but usually with modest power. Thus, system design and engineering issues are analyzed for approaches and trade-offs that can make the best use of semiconductor laser capabilities in multispectral imaging. A few systems were developed and the technique was tested and evaluated on a variety of natural and man-made objects. It was shown capable of high spectral resolution imaging which, unlike non-imaging point sensing, allows detecting and discriminating objects of interest even without a priori spectroscopic knowledge of the targets. Examples include material and chemical discrimination. It was also shown capable of dealing with the complexity of interpreting diffuse scattered spectral images and produced results that could otherwise be ambiguous with conventional imaging. Examples with glucose and spectral imaging of drug pills were discussed. Lastly, the technique was shown with conventional laser spectroscopy such as wavelength modulation spectroscopy to image a gas (CO). These results suggest the versatility and power of multi-spectral laser imaging, which can be practical with the use of semiconductor lasers.

  16. Semiconductor Laser Multi-Spectral Sensing and Imaging

    PubMed Central

    Le, Han Q.; Wang, Yang

    2010-01-01

    Multi-spectral laser imaging is a technique that can offer a combination of the laser capability of accurate spectral sensing with the desirable features of passive multispectral imaging. The technique can be used for detection, discrimination, and identification of objects by their spectral signature. This article describes and reviews the development and evaluation of semiconductor multi-spectral laser imaging systems. Although the method is certainly not specific to any laser technology, the use of semiconductor lasers is significant with respect to practicality and affordability. More relevantly, semiconductor lasers have their own characteristics; they offer excellent wavelength diversity but usually with modest power. Thus, system design and engineering issues are analyzed for approaches and trade-offs that can make the best use of semiconductor laser capabilities in multispectral imaging. A few systems were developed and the technique was tested and evaluated on a variety of natural and man-made objects. It was shown capable of high spectral resolution imaging which, unlike non-imaging point sensing, allows detecting and discriminating objects of interest even without a priori spectroscopic knowledge of the targets. Examples include material and chemical discrimination. It was also shown capable of dealing with the complexity of interpreting diffuse scattered spectral images and produced results that could otherwise be ambiguous with conventional imaging. Examples with glucose and spectral imaging of drug pills were discussed. Lastly, the technique was shown with conventional laser spectroscopy such as wavelength modulation spectroscopy to image a gas (CO). These results suggest the versatility and power of multi-spectral laser imaging, which can be practical with the use of semiconductor lasers. PMID:22315555

  17. Multiplexed Spectral Imaging of 120 Different Fluorescent Labels

    PubMed Central

    Valm, Alex M.; Oldenbourg, Rudolf; Borisy, Gary G.

    2016-01-01

    The number of fluorescent labels that can unambiguously be distinguished in a single image when acquired through band pass filters is severely limited by the spectral overlap of available fluorophores. The recent development of spectral microscopy and the application of linear unmixing algorithms to spectrally recorded image data have allowed simultaneous imaging of fluorophores with highly overlapping spectra. However, the number of distinguishable fluorophores is still limited by the unavoidable decrease in signal to noise ratio when fluorescence signals are fractionated over multiple wavelength bins. Here we present a spectral image analysis algorithm to greatly expand the number of distinguishable objects labeled with binary combinations of fluorophores. Our algorithm utilizes a priori knowledge about labeled specimens and imposes a binary label constraint on the unmixing solution. We have applied our labeling and analysis strategy to identify microbes labeled by fluorescence in situ hybridization and here demonstrate the ability to distinguish 120 differently labeled microbes in a single image. PMID:27391327

  18. [Research on Spectral Polarization Imaging System Based on Static Modulation].

    PubMed

    Zhao, Hai-bo; Li, Huan; Lin, Xu-ling; Wang, Zheng

    2015-04-01

    The main disadvantages of traditional spectral polarization imaging system are: complex structure, with moving parts, low throughput. A novel method of spectral polarization imaging system is discussed, which is based on static polarization intensity modulation combined with Savart polariscope interference imaging. The imaging system can obtain real-time information of spectral and four Stokes polarization messages. Compared with the conventional methods, the advantages of the imaging system are compactness, low mass and no moving parts, no electrical control, no slit and big throughput. The system structure and the basic theory are introduced. The experimental system is established in the laboratory. The experimental system consists of reimaging optics, polarization intensity module, interference imaging module, and CCD data collecting and processing module. The spectral range is visible and near-infrared (480-950 nm). The white board and the plane toy are imaged by using the experimental system. The ability of obtaining spectral polarization imaging information is verified. The calibration system of static polarization modulation is set up. The statistical error of polarization degree detection is less than 5%. The validity and feasibility of the basic principle is proved by the experimental result. The spectral polarization data captured by the system can be applied to object identification, object classification and remote sensing detection.

  19. Volumetric sub-surface imaging using spectrally encoded endoscopy.

    PubMed

    Yelin, D; Bouma, B E; Tearney, G J

    2008-02-04

    Endoscopic imaging below tissue surfaces and through turbid media may provide improved diagnostic capabilities and visibility in surgical settings. Spectrally encoded endoscopy (SEE) is a recently developed method that utilizes a single optical fiber, miniature optics and a diffractive grating for high-speed imaging through small diameter, flexible endoscopic probes. SEE has also been shown to provide three-dimensional topological imaging capabilities. In this paper, we have configured SEE to additionally image beneath tissue surfaces, by increasing the system's sensitivity and acquiring the complex spectral density for each spectrally resolved point on the sample. In order to demonstrate the capability of SEE to obtain subsurface information, we have utilized the system to image a resolution target through intralipid solution, and conduct volumetric imaging of a mouse embryo and excised human middle-ear ossicles. Our results demonstrate that real-time subsurface imaging is possible with this miniature endoscopy technique.

  20. Local Spectral Component Decomposition for Multi-Channel Image Denoising.

    PubMed

    Rizkinia, Mia; Baba, Tatsuya; Shirai, Keiichiro; Okuda, Masahiro

    2016-07-01

    We propose a method for local spectral component decomposition based on the line feature of local distribution. Our aim is to reduce noise on multi-channel images by exploiting the linear correlation in the spectral domain of a local region. We first calculate a linear feature over the spectral components of an M -channel image, which we call the spectral line, and then, using the line, we decompose the image into three components: a single M -channel image and two gray-scale images. By virtue of the decomposition, the noise is concentrated on the two images, and thus our algorithm needs to denoise only the two gray-scale images, regardless of the number of the channels. As a result, image deterioration due to the imbalance of the spectral component correlation can be avoided. The experiment shows that our method improves image quality with less deterioration while preserving vivid contrast. Our method is especially effective for hyperspectral images. The experimental results demonstrate that our proposed method can compete with the other state-of-the-art denoising methods.

  1. A velocity-map imaging study of methyl non-resonant multiphoton ionization from the photodissociation of CH3I in the A-band.

    PubMed

    Poullain, Sonia Marggi; Chicharro, David V; Rubio-Lago, Luis; García-Vela, Alberto; Bañares, Luis

    2017-04-28

    Chemical reaction dynamics and, particularly, photodissociation in the gas phase are generally studied using pump-probe schemes where a first laser pulse induces the process under study and a second one detects the produced fragments. Providing an efficient detection of ro-vibrationally state-selected photofragments, the resonance enhanced multiphoton ionization (REMPI) technique is, without question, the most popular approach used for the probe step, while non-resonant multiphoton ionization (NRMPI) detection of the products is scarce. The main goal of this work is to test the sensitivity of the NRMPI technique to fragment vibrational distributions arising from molecular photodissociation processes. We revisit the well-known process of methyl iodide photodissociation in the A-band at around 280 nm, using the velocity-map imaging technique in conjunction with NRMPI of the methyl fragment. The detection wavelength, carefully selected to avoid any REMPI transition, was scanned between 325 and 335 nm seeking correlations between the different observables-the product vibrational, translational and angular distributions-and the excitation wavelength of the probe laser pulse. The experimental results have been discussed on the base of quantum dynamics calculations of photofragment vibrational populations carried out on available ab initio potential-energy surfaces using a four-dimensional model.This article is part of the themed issue 'Theoretical and computational studies of non-equilibrium and non-statistical dynamics in the gas phase, in the condensed phase and at interfaces'.

  2. Tri-modal microscopy with multiphoton and optical coherence microscopy/tomography for multi-scale and multi-contrast imaging

    PubMed Central

    Chong, Shau Poh; Lai, Tom; Zhou, Yifeng; Tang, Shuo

    2013-01-01

    Multi-scale multimodal microscopy is a very useful technique by providing multiple imaging contrasts with adjustable field of views and spatial resolutions. Here, we present a tri-modal microscope combining multiphoton microscopy (MPM), optical coherence microscopy (OCM) and optical coherence tomography (OCT) for subsurface visualization of biological tissues. The advantages of the tri-modal system are demonstrated on various biological samples. It enables the visualization of multiple intrinsic contrasts including scattering, two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG). It also enables a rapid scanning over a large tissue area and a high resolution zoom-in for cellular-level structures on regions of interest. The tri-modal microscope can be important for label-free imaging to obtain a sufficient set of parameters for reliable sample analysis. PMID:24049679

  3. Multiphoton microscopy in defining liver function

    NASA Astrophysics Data System (ADS)

    Thorling, Camilla A.; Crawford, Darrell; Burczynski, Frank J.; Liu, Xin; Liau, Ian; Roberts, Michael S.

    2014-09-01

    Multiphoton microscopy is the preferred method when in vivo deep-tissue imaging is required. This review presents the application of multiphoton microscopy in defining liver function. In particular, multiphoton microscopy is useful in imaging intracellular events, such as mitochondrial depolarization and cellular metabolism in terms of NAD(P)H changes with fluorescence lifetime imaging microscopy. The morphology of hepatocytes can be visualized without exogenously administered fluorescent dyes by utilizing their autofluorescence and second harmonic generation signal of collagen, which is useful in diagnosing liver disease. More specific imaging, such as studying drug transport in normal and diseased livers are achievable, but require exogenously administered fluorescent dyes. If these techniques can be translated into clinical use to assess liver function, it would greatly improve early diagnosis of organ viability, fibrosis, and cancer.

  4. Spectral reflectance and digital image relations among five aquatic weeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study reports on the use of an artificial quartz halogen lighting source to facilitate the acquisition of spectral light reflectance measurements and digital imaging of invasive aquatic weeds. Spectral leaf or leaf/stem reflectance measurements were made on five aquatic weeds: Eurasian watermil...

  5. New spectral imaging techniques for blood oximetry in the retina

    NASA Astrophysics Data System (ADS)

    Alabboud, Ied; Muyo, Gonzalo; Gorman, Alistair; Mordant, David; McNaught, Andrew; Petres, Clement; Petillot, Yvan R.; Harvey, Andrew R.

    2007-07-01

    Hyperspectral imaging of the retina presents a unique opportunity for direct and quantitative mapping of retinal biochemistry - particularly of the vasculature where blood oximetry is enabled by the strong variation of absorption spectra with oxygenation. This is particularly pertinent both to research and to clinical investigation and diagnosis of retinal diseases such as diabetes, glaucoma and age-related macular degeneration. The optimal exploitation of hyperspectral imaging however, presents a set of challenging problems, including; the poorly characterised and controlled optical environment of structures within the retina to be imaged; the erratic motion of the eye ball; and the compounding effects of the optical sensitivity of the retina and the low numerical aperture of the eye. We have developed two spectral imaging techniques to address these issues. We describe first a system in which a liquid crystal tuneable filter is integrated into the illumination system of a conventional fundus camera to enable time-sequential, random access recording of narrow-band spectral images. Image processing techniques are described to eradicate the artefacts that may be introduced by time-sequential imaging. In addition we describe a unique snapshot spectral imaging technique dubbed IRIS that employs polarising interferometry and Wollaston prism beam splitters to simultaneously replicate and spectrally filter images of the retina into multiple spectral bands onto a single detector array. Results of early clinical trials acquired with these two techniques together with a physical model which enables oximetry map are reported.

  6. Multimodal multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Légaré, François; Pfeffer, Christian P.; Ganikhanov, Feruz

    2009-02-01

    Multiphoton microscopy is a powerful technique for high spatial resolution thick tissue imaging. In its simple version, it uses a high repetition rate femtosecond oscillator laser source focussed and scanned across biological sample that contains fluorophores. However, not every biological structure is inherently fluorescent or can be stained without causing biochemical changes. To circumvent these limitations, other non-invasive nonlinear optical imaging approaches are currently being developed and investigated with regard to different applications. These techniques are: (1) second harmonic generation (SHG), (2) third harmonic generation (THG), and (3) coherent anti-Stokes Raman scattering (CARS) microscopy. The main advantage of the above mentioned techniques is that they derive their imaging contrast from optical nonlinearities that do not involve fluorescence process. As a particular application example we investigated collagen arrays. We show that combining SHG-THG-CARS onto a single imaging platform provides complementary information about the sub-micron architecture of the tissue. SHG microscopy reveals the fibrillar architecture of collagen arrays and confirm a rather high degree of heterogeneity of χ(2) within the focal volume, THG highlights the boundaries between the collagen sheets, and CH2 spectroscopic contrast with CARS.

  7. Spectral characteristics of two-photon autofluorescence and second harmonic generation from human skin in vivo

    NASA Astrophysics Data System (ADS)

    Breunig, Hans G.; König, Karsten

    2011-03-01

    We performed multiphoton imaging of human skin and recorded in combination the complete spectral content of the signals in vivo. The spectra represent the integration of multiphoton signals over the investigated regions of the epidermis and dermis. They are used to study depth-resolved in vivo emission characteristics of main endogenous skin fluorophores like keratin, NAD(P)H, collagen and elastin. The identification of the specific fluorophores is supported by analysis of additional in vivo fluorescence lifetime imaging. Furthermore, as a potential application of spectrally selective imaging the possibility to investigate the penetration of nanoparticles from sunscreen lotion into skin in vivo is discussed.

  8. Retinex Preprocessing for Improved Multi-Spectral Image Classification

    NASA Technical Reports Server (NTRS)

    Thompson, B.; Rahman, Z.; Park, S.

    2000-01-01

    The goal of multi-image classification is to identify and label "similar regions" within a scene. The ability to correctly classify a remotely sensed multi-image of a scene is affected by the ability of the classification process to adequately compensate for the effects of atmospheric variations and sensor anomalies. Better classification may be obtained if the multi-image is preprocessed before classification, so as to reduce the adverse effects of image formation. In this paper, we discuss the overall impact on multi-spectral image classification when the retinex image enhancement algorithm is used to preprocess multi-spectral images. The retinex is a multi-purpose image enhancement algorithm that performs dynamic range compression, reduces the dependence on lighting conditions, and generally enhances apparent spatial resolution. The retinex has been successfully applied to the enhancement of many different types of grayscale and color images. We show in this paper that retinex preprocessing improves the spatial structure of multi-spectral images and thus provides better within-class variations than would otherwise be obtained without the preprocessing. For a series of multi-spectral images obtained with diffuse and direct lighting, we show that without retinex preprocessing the class spectral signatures vary substantially with the lighting conditions. Whereas multi-dimensional clustering without preprocessing produced one-class homogeneous regions, the classification on the preprocessed images produced multi-class non-homogeneous regions. This lack of homogeneity is explained by the interaction between different agronomic treatments applied to the regions: the preprocessed images are closer to ground truth. The principle advantage that the retinex offers is that for different lighting conditions classifications derived from the retinex preprocessed images look remarkably "similar", and thus more consistent, whereas classifications derived from the original

  9. A parametric estimation approach to instantaneous spectral imaging.

    PubMed

    Oktem, Figen S; Kamalabadi, Farzad; Davila, Joseph M

    2014-12-01

    Spectral imaging, the simultaneous imaging and spectroscopy of a radiating scene, is a fundamental diagnostic technique in the physical sciences with widespread application. Due to the intrinsic limitation of two-dimensional (2D) detectors in capturing inherently three-dimensional (3D) data, spectral imaging techniques conventionally rely on a spatial or spectral scanning process, which renders them unsuitable for dynamic scenes. In this paper, we present a nonscanning (instantaneous) spectral imaging technique that estimates the physical parameters of interest by combining measurements with a parametric model and solving the resultant inverse problem computationally. The associated inverse problem, which can be viewed as a multiframe semiblind deblurring problem (with shift-variant blur), is formulated as a maximum a posteriori (MAP) estimation problem since in many such experiments prior statistical knowledge of the physical parameters can be well estimated. Subsequently, an efficient dynamic programming algorithm is developed to find the global optimum of the nonconvex MAP problem. Finally, the algorithm and the effectiveness of the spectral imaging technique are illustrated for an application in solar spectral imaging. Numerical simulation results indicate that the physical parameters can be estimated with the same order of accuracy as state-of-the-art slit spectroscopy but with the added benefit of an instantaneous, 2D field-of-view. This technique will be particularly useful for studying the spectra of dynamic scenes encountered in space remote sensing.

  10. Snapshot spectral and polarimetric imaging; target identification with multispectral video

    NASA Astrophysics Data System (ADS)

    Bartlett, Brent D.; Rodriguez, Mikel D.

    2013-05-01

    As the number of pixels continue to grow in consumer and scientific imaging devices, it has become feasible to collect the incident light field. In this paper, an imaging device developed around light field imaging is used to collect multispectral and polarimetric imagery in a snapshot fashion. The sensor is described and a video data set is shown highlighting the advantage of snapshot spectral imaging. Several novel computer vision approaches are applied to the video cubes to perform scene characterization and target identification. It is shown how the addition of spectral and polarimetric data to the video stream allows for multi-target identification and tracking not possible with traditional RGB video collection.

  11. Application of coherent antistokes Raman scattering (CARS) to imaging mammalian cells: a means for gaining molecular selectivity in multiphoton imaging

    NASA Astrophysics Data System (ADS)

    Holtom, Gary R.; Thrall, Brian; Weber, Thomas; Zhu, Leyun; Hopkins, Derek; Parkinson, Christopher; Colson, Steven; Price, John M.; Chin, Beek Yoke; Choi, Augustine; Risby, Terence

    2001-04-01

    Virtually all laser based microscopy imaging methods involve a single laser, with ultrafast lasers emerging as the enabling tool for a variety of methods. Two-photon fluorescence is a high sensitivity method with selectivity depending on a chromophore that is either added or produced by genetic engineering. While there are fundamental advantages over white light or other fluorescence microscopies, there are unavoidable limitations such as bleaching, photoinduced damage to the cell, and the inability to label some major constituents of the cell, particularly the abundant species. Raman imaging affords chemical selectivity but application is limited due particularly to its low sensitivity and unavoidable fluorescence background. Adding a second laser beam, shifted from the first laser by a molecular vibrational frequency, increases the detected Raman signal by many orders of magnitude and in addition shifts the detected signal to the high energy (blue) side of both lasers, removing fluorescence artifacts. Signal levels sufficient to acquire high signal-to-noise ratio images of 200 by 200 pixels in one minute requires sub-nanojoule pulse energy. A convenient, tunable source of the Stokes-shifted beam is provided by an Optical Parametric Amplifier (OPA), which requires an amplified laser. 250-kHz sources have ample energy and in addition keep the average sample power on the order of 0.1 mW, a level that even sensitive biological systems tolerate at the focal spot diameter of 0.3 micrometers . Long-term viability of mammalian cells has been demonstrated during dozens of scans in a single plane. Two-photon fluorescence provides a useful complimentary data channel that is acquired simultaneously with the Raman image. Several dyes and green fluorescence protein have been used for this purpose. Interpretation of images, acquiring three dimensional images, and identification of cellular features are ongoing activities.

  12. Integrated filter and detector array for spectral imaging

    NASA Technical Reports Server (NTRS)

    Labaw, Clayton C. (Inventor)

    1992-01-01

    A spectral imaging system having an integrated filter and photodetector array is disclosed. The filter has narrow transmission bands which vary in frequency along the photodetector array. The frequency variation of the transmission bands is matched to, and aligned with, the frequency variation of a received spectral image. The filter is deposited directly on the photodetector array by a low temperature deposition process. By depositing the filter directly on the photodetector array, permanent alignment is achieved for all temperatures, spectral crosstalk is substantially eliminated, and a high signal to noise ratio is achieved.

  13. Multilayer structure for a spectral imaging sensor.

    PubMed

    Parrein, Pascale; Moussy, Norbert; Poupinet, Ludovic; Gidon, Pierre

    2009-01-20

    We investigate the possibility of recovering spectral information using a multilayer structure realized through microelectronics technologies and compatible with a matrix arrangement. The structure is made of photoabsorbing layers, acting as local photodetectors, alternating with transparent layers. The whole structure lies on a reflective surface. A stationary wave containing the spectral information of the source is generated within the structure. We determine the intensity of the stationary wave at any position, taking into account absorption and multireflections at each transition as well as the signal detected by the photoabsorbing layers. The model forecasting the detected signal is then validated using p-i-n diodes of different thicknesses made of hydrogenated amorphous silicon (a-Si:H) encompassed between indium tin oxide (ITO) electrodes. The detected signal depends on the wavelength of the incident light, the thickness of the detecting layer, and the latter's position within the structure. A specific spectral response can then be associated to each photoabsorbing layer. We show how spectral information can be retrieved from this kind of structure in the visible spectrum range.

  14. Some simple mechanisms of multiphoton excitation in many - level systems

    NASA Astrophysics Data System (ADS)

    Donley, E. A.; Marquardt, R.; Quack, M.; Stohner, J.; Thanopulos, I.; Wallenborn, E.-U.

    Results are reported on coherent monochromatic multiphoton excitation in many-level systems, which are representative for some of the basic mechanisms for atomic and molecular multiphoton processes. Numerical solutions are discussed that use the Floquet and quasiresonant approximations in the framework of the URIMIR program package. The excitation schemes include direct three-photon excitation, two-photon excitation with diagonal coupling, Göppert-Mayer-type two-photon processes, multiphoton excitation with off-resonant intermediates, and practically irreversible coherent excitation into dense spectral structures. Several interesting phenomena are observed, such as nonlinear line shifts and broadenings of multiphoton resonances of relevance for multiphoton spectroscopy and almost constant intermediate population inversions, potentially useful for laser design. The accurate numerical results are compared with approximate solutions from perturbation theory, and with simple analytical solutions from Rabi-type formulae.

  15. Unsupervised texture image segmentation using multilayer data condensation spectral clustering

    NASA Astrophysics Data System (ADS)

    Liu, Hanqiang; Jiao, Licheng; Zhao, Feng

    2010-07-01

    A novel unsupervised texture image segmentation using a multilayer data condensation spectral clustering algorithm is presented. First, the texture features of each image pixel are extracted by the stationary wavelet transform and a multilayer data condensation method is performed on this texture features data set to obtain a condensation subset. Second, the spectral clustering algorithm based on the manifold similarity measure is used to cluster the condensation subset. Finally, according to the clustering result of the condensation subset, the nearest-neighbor method is adopted to obtain the original image-segmentation result. In the experiments, we apply our method to solve the texture and synthetic aperture radar image segmentation and take self-tuning k-nearest-neighbor spectral clustering and Nyström methods for baseline comparisons. The experimental results show that the proposed method is more robust and effective for texture image segmentation.

  16. Hyper-spectral image segmentation using spectral clustering with covariance descriptors

    NASA Astrophysics Data System (ADS)

    Kursun, Olcay; Karabiber, Fethullah; Koc, Cemalettin; Bal, Abdullah

    2009-02-01

    Image segmentation is an important and difficult computer vision problem. Hyper-spectral images pose even more difficulty due to their high-dimensionality. Spectral clustering (SC) is a recently popular clustering/segmentation algorithm. In general, SC lifts the data to a high dimensional space, also known as the kernel trick, then derive eigenvectors in this new space, and finally using these new dimensions partition the data into clusters. We demonstrate that SC works efficiently when combined with covariance descriptors that can be used to assess pixelwise similarities rather than in the high-dimensional Euclidean space. We present the formulations and some preliminary results of the proposed hybrid image segmentation method for hyper-spectral images.

  17. Spectral image reconstruction by a tunable LED illumination

    NASA Astrophysics Data System (ADS)

    Lin, Meng-Chieh; Tsai, Chen-Wei; Tien, Chung-Hao

    2013-09-01

    Spectral reflectance estimation of an object via low-dimensional snapshot requires both image acquisition and a post numerical estimation analysis. In this study, we set up a system incorporating a homemade cluster of LEDs with spectral modulation for scene illumination, and a multi-channel CCD to acquire multichannel images by means of fully digital process. Principal component analysis (PCA) and pseudo inverse transformation were used to reconstruct the spectral reflectance in a constrained training set, such as Munsell and Macbeth Color Checker. The average reflectance spectral RMS error from 34 patches of a standard color checker were 0.234. The purpose is to investigate the use of system in conjunction with the imaging analysis for industry or medical inspection in a fast and acceptable accuracy, where the approach was preliminary validated.

  18. Methods for spectral image analysis by exploiting spatial simplicity

    DOEpatents

    Keenan, Michael R.

    2010-11-23

    Several full-spectrum imaging techniques have been introduced in recent years that promise to provide rapid and comprehensive chemical characterization of complex samples. One of the remaining obstacles to adopting these techniques for routine use is the difficulty of reducing the vast quantities of raw spectral data to meaningful chemical information. Multivariate factor analysis techniques, such as Principal Component Analysis and Alternating Least Squares-based Multivariate Curve Resolution, have proven effective for extracting the essential chemical information from high dimensional spectral image data sets into a limited number of components that describe the spectral characteristics and spatial distributions of the chemical species comprising the sample. There are many cases, however, in which those constraints are not effective and where alternative approaches may provide new analytical insights. For many cases of practical importance, imaged samples are "simple" in the sense that they consist of relatively discrete chemical phases. That is, at any given location, only one or a few of the chemical species comprising the entire sample have non-zero concentrations. The methods of spectral image analysis of the present invention exploit this simplicity in the spatial domain to make the resulting factor models more realistic. Therefore, more physically accurate and interpretable spectral and abundance components can be extracted from spectral images that have spatially simple structure.

  19. Methods for spectral image analysis by exploiting spatial simplicity

    DOEpatents

    Keenan, Michael R.

    2010-05-25

    Several full-spectrum imaging techniques have been introduced in recent years that promise to provide rapid and comprehensive chemical characterization of complex samples. One of the remaining obstacles to adopting these techniques for routine use is the difficulty of reducing the vast quantities of raw spectral data to meaningful chemical information. Multivariate factor analysis techniques, such as Principal Component Analysis and Alternating Least Squares-based Multivariate Curve Resolution, have proven effective for extracting the essential chemical information from high dimensional spectral image data sets into a limited number of components that describe the spectral characteristics and spatial distributions of the chemical species comprising the sample. There are many cases, however, in which those constraints are not effective and where alternative approaches may provide new analytical insights. For many cases of practical importance, imaged samples are "simple" in the sense that they consist of relatively discrete chemical phases. That is, at any given location, only one or a few of the chemical species comprising the entire sample have non-zero concentrations. The methods of spectral image analysis of the present invention exploit this simplicity in the spatial domain to make the resulting factor models more realistic. Therefore, more physically accurate and interpretable spectral and abundance components can be extracted from spectral images that have spatially simple structure.

  20. Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

    NASA Astrophysics Data System (ADS)

    Darvin, M. E.; Richter, H.; Zhu, Y. J.; Meinke, M. C.; Knorr, F.; Gonchukov, S. A.; Koenig, K.; Lademann, J.

    2014-07-01

    Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted.

  1. Comparison of in vivo and ex vivo laser scanning microscopy and multiphoton tomography application for human and porcine skin imaging

    SciTech Connect

    Darvin, M E; Richter, H; Zhu, Y J; Meinke, M C; Knorr, F; Lademann, J; Gonchukov, S A; Koenig, K

    2014-07-31

    Two state-of-the-art microscopic optical methods, namely, confocal laser scanning microscopy in the fluorescence and reflectance regimes and multiphoton tomography in the autofluorescence and second harmonic generation regimes, are compared for porcine skin ex vivo and healthy human skin in vivo. All skin layers such as stratum corneum (SC), stratum spinosum (SS), stratum basale (SB), papillary dermis (PD) and reticular dermis (RD) as well as transition zones between these skin layers are measured noninvasively at a high resolution, using the above mentioned microscopic methods. In the case of confocal laser scanning microscopy (CLSM), measurements in the fluorescence regime were performed by using a fluorescent dye whose topical application on the surface is well suited for the investigation of superficial SC and characterisation of the skin barrier function. For investigations of deeply located skin layers, such as SS, SB and PD, the fluorescent dye must be injected into the skin, which markedly limits fluorescence measurements using CLSM. In the case of reflection CLSM measurements, the obtained results can be compared to the results of multiphoton tomography (MPT) for all skin layers excluding RD. CLSM cannot distinguish between dermal collagen and elastin measuring their superposition in the RD. By using MPT, it is possible to analyse the collagen and elastin structures separately, which is important for the investigation of anti-aging processes. The resolution of MPT is superior to CLSM. The advantages and limitations of both methods are discussed and the differences and similarities between human and porcine skin are highlighted. (laser biophotonics)

  2. Research on method of infrared spectral imaging based on thermal imager

    NASA Astrophysics Data System (ADS)

    Huan, Ke-wei; Shi, Xiao-guang; Wu, Wei; Zheng, Feng; Liu, Xiao-xi

    2011-08-01

    In recent years, technology of thermal imager and spectral imaging is becoming mature, and the application of them is increased. The method is based on the blackbody radiation theory, make use of the infrared thermal imager to collect and analysis the thermal images, distill the temperature value of different pixel of the thermal images, use Matlab to deal blackbody radiation emitted curve fitting according with the temperature value of different pixels, and get the values of the degree of radiation emitted at the same wavelength from the different pixels, then make spectral imaging (1μm~10μm) according to the values. At last, do analysis to spectral imaging of different spectral bands; discuss the limitations of using this method to achieve spectral imaging.

  3. Advances in Spectral-Spatial Classification of Hyperspectral Images

    NASA Technical Reports Server (NTRS)

    Fauvel, Mathieu; Tarabalka, Yuliya; Benediktsson, Jon Atli; Chanussot, Jocelyn; Tilton, James C.

    2012-01-01

    Recent advances in spectral-spatial classification of hyperspectral images are presented in this paper. Several techniques are investigated for combining both spatial and spectral information. Spatial information is extracted at the object (set of pixels) level rather than at the conventional pixel level. Mathematical morphology is first used to derive the morphological profile of the image, which includes characteristics about the size, orientation and contrast of the spatial structures present in the image. Then the morphological neighborhood is defined and used to derive additional features for classification. Classification is performed with support vector machines using the available spectral information and the extracted spatial information. Spatial post-processing is next investigated to build more homogeneous and spatially consistent thematic maps. To that end, three presegmentation techniques are applied to define regions that are used to regularize the preliminary pixel-wise thematic map. Finally, a multiple classifier system is defined to produce relevant markers that are exploited to segment the hyperspectral image with the minimum spanning forest algorithm. Experimental results conducted on three real hyperspectral images with different spatial and spectral resolutions and corresponding to various contexts are presented. They highlight the importance of spectral-spatial strategies for the accurate classification of hyperspectral images and validate the proposed methods.

  4. Fast DPCM scheme for lossless compression of aurora spectral images

    NASA Astrophysics Data System (ADS)

    Kong, Wanqiu; Wu, Jiaji

    2016-10-01

    Aurora has abundant information to be stored. Aurora spectral image electronically preserves spectral information and visual observation of aurora during a period to be studied later. These images are helpful for the research of earth-solar activities and to understand the aurora phenomenon itself. However, the images are produced with a quite high sampling frequency, which leads to the challenging transmission load. In order to solve the problem, lossless compression turns out to be required. Indeed, each frame of aurora spectral images differs from the classical natural image and also from the frame of hyperspectral image. Existing lossless compression algorithms are not quite applicable. On the other hand, the key of compression is to decorrelate between pixels. We consider exploiting a DPCM-based scheme for the lossless compression because DPCM is effective for decorrelation. Such scheme makes use of two-dimensional redundancy both in the spatial and spectral domain with a relatively low complexity. Besides, we also parallel it for a faster computation speed. All codes are implemented on a structure consists of nested for loops of which the outer and the inner loops are respectively designed for spectral and spatial decorrelation. And the parallel version is represented on CPU platform using different numbers of cores. Experimental results show that compared to traditional lossless compression methods, the DPCM scheme has great advantage in compression gain and meets the requirement of real-time transmission. Besides, the parallel version has expected computation performance with a high CPU utilization.

  5. Multi-spectral confocal microendoscope for in-vivo imaging

    NASA Astrophysics Data System (ADS)

    Rouse, Andrew Robert

    The concept of in-vivo multi-spectral confocal microscopy is introduced. A slit-scanning multi-spectral confocal microendoscope (MCME) was built to demonstrate the technique. The MCME employs a flexible fiber-optic catheter coupled to a custom built slit-scan confocal microscope fitted with a custom built imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The design and performance of the miniature objective and focus assembly are discussed. The 3mm diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope. The confocal nature of the system provides optical sectioning with 3mum lateral resolution and 30mum axial resolution. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible chromatic range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 7nm to 18nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersive power of the prism. The MCME is designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. The primary applications for the system include diagnosis of disease in the gastro-intestinal tract and female reproductive system. Recent data from the grayscale imaging mode are presented. Preliminary multi-spectral results from phantoms, cell cultures, and excised human tissue are presented to demonstrate the potential of in-vivo multi-spectral imaging.

  6. Interactive image segmentation by constrained spectral graph partitioning

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; He, Jin; Zhang, Hong; Huang, Zhanhua

    2010-11-01

    This paper proposed an interactive image segmentation algorithm that can tolerate slightly incorrect user constraints. Interactive image segmentation was formulated as a constrained spectral graph partitioning problem. Furthermore, it was proven to equal to a supervised classification problem, where the feature space was formed by rows of the eigenvector matrix that was computed by spectral graph analysis. ν-SVM (support vector machine) was preferred as the classifier. Some incorrect labels in user constraints were tolerated by being identified as margin errors in ν-SVM. Comparison with other algorithms on real color images was reported.

  7. Motion correction for cellular-resolution multi-photon fluorescence microscopy imaging of awake head-restrained mice using speed embedded HMM.

    PubMed

    Chen, Taoyi; Xue, Zhong; Wang, Changhong; Qu, Zhenshen; Wong, Kelvin K; Wong, Stephen T C

    2012-04-01

    Multi-photon fluorescence microscopy (MFM) captures high-resolution fluorescence image sequences and can be used for the intact brain imaging of small animals. Recently, it has been extended from anesthetized and head-stabilized mice to awake and head-restrained ones for in vivo neurological study. In these applications, motion correction is an important pre-processing step since brain pulsation and body movement can cause motion artifact and prevent stable serial image acquisition at such high spatial resolution. This paper proposes a speed embedded Hidden Markov model (SEHMM) for motion correction in MFM imaging of awake head-restrained mice. The algorithm extends the traditional Hidden Markov model (HMM) method by embedding a motion prediction model to better estimate the state transition probability. The novelty of the method lies in using adaptive probability to estimate the linear motion, while the state-of-the-art method assumes that the highest probability is assigned to the case with no motion. In experiments we demonstrated that SEHMM is more accurate than the traditional HMM using both simulated and real MFM image sequences.

  8. Multi-spectral imaging with mid-infrared semiconductor lasers

    NASA Astrophysics Data System (ADS)

    Wang, Yi; Wang, Yang; Le, Han Q.

    2006-01-01

    Multi-spectral laser imaging can be a useful technology for target discrimination, classification, and identification based on object spectral signatures. The mid-IR region (~3-14 μm) is particularly rich of molecular spectroscopic fingerprints, but the technology has been under utilized. Compact, potentially inexpensive semiconductor lasers may allow more cost-effective applications. This paper describes a development of semiconductor-laser-based multi-spectral imaging for both near-IR and mid-IR, and demonstrates the potential of this technology. The near-IR study employed 7 wavelengths from 0.635-1.55 μm, and used for system engineering evaluation as well as for studying the fundamental aspects of multi-spectral laser imaging. These include issues of wavelength-dependence scattering as a function of incident and receiving angle and the polarization effects. Stokes vector imaging and degree-of-linear-polarization were shown to reveal significant information to characterize the targets. The mid-IR study employed 4 wavelengths from 3.3-9.6 μm, and was applied to diverse targets that consist of natural and man-made materials and household objects. It was shown capable to resolve and distinguish small spectral differences among various targets, thanks to the laser radiometric and spectral accuracy. Colorless objects in the visible were shown with "colorful" signatures in the mid-IR. An essential feature of the study is an advanced system architecture that employs wavelength-division-multiplexed laser beams for high spectral fidelity and resolution. In addition, unlike conventional one-transmitter and one receiver design, the system is based on a scalable CDMA network concept with multiple transmitters and receivers to allow efficient information acquisition. The results suggest that multi-spectral laser imaging in general can be a unique and powerful technology for wide ranging applications.

  9. Design and development of compact multiphoton microscopes

    NASA Astrophysics Data System (ADS)

    Mehravar, SeyedSoroush

    A compact multi-photon microscope (MPM) was designed and developed with the use of low-cost mode-locked fiber lasers operating at 1040nm and 1560nm. The MPM was assembled in-house and the system aberration was investigated using the optical design software: Zemax. A novel characterization methodology based on 'nonlinear knife-edge' technique was also introduced to measure the axial, lateral resolution, and the field curvature of the multi-photon microscope's image plane. The field curvature was then post-corrected using data processing in MATLAB. A customized laser scanning software based on LabVIEW was developed for data acquisition, image display and controlling peripheral electronics. Finally, different modalities of multi-photon excitation such as second- and third harmonic generation, two- and three-photon fluorescence were utilized to study a wide variety of samples from cancerous cells to 2D-layered materials.

  10. A pragmatic guide to multiphoton microscope design

    PubMed Central

    Young, Michael D.; Field, Jeffrey J.; Sheetz, Kraig E.; Bartels, Randy A.; Squier, Jeff

    2016-01-01

    Multiphoton microscopy has emerged as a ubiquitous tool for studying microscopic structure and function across a broad range of disciplines. As such, the intent of this paper is to present a comprehensive resource for the construction and performance evaluation of a multiphoton microscope that will be understandable to the broad range of scientific fields that presently exploit, or wish to begin exploiting, this powerful technology. With this in mind, we have developed a guide to aid in the design of a multiphoton microscope. We discuss source selection, optical management of dispersion, image-relay systems with scan optics, objective-lens selection, single-element light-collection theory, photon-counting detection, image rendering, and finally, an illustrated guide for building an example microscope. PMID:27182429

  11. Rank minimization code aperture design for spectrally selective compressive imaging.

    PubMed

    Arguello, Henry; Arce, Gonzalo R

    2013-03-01

    A new code aperture design framework for multiframe code aperture snapshot spectral imaging (CASSI) system is presented. It aims at the optimization of code aperture sets such that a group of compressive spectral measurements is constructed, each with information from a specific subset of bands. A matrix representation of CASSI is introduced that permits the optimization of spectrally selective code aperture sets. Furthermore, each code aperture set forms a matrix such that rank minimization is used to reduce the number of CASSI shots needed. Conditions for the code apertures are identified such that a restricted isometry property in the CASSI compressive measurements is satisfied with higher probability. Simulations show higher quality of spectral image reconstruction than that attained by systems using Hadamard or random code aperture sets.

  12. Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts

    PubMed Central

    Bardet, Sylvia M.; Carr, Lynn; Soueid, Malak; Arnaud-Cormos, Delia; Leveque, Philippe; O’Connor, Rodney P.

    2016-01-01

    Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35–45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma. PMID:27698479

  13. Control of multiphoton molecular excitation with shaped femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Xu, Bingwei

    The work presented in this dissertation describes the use of shaped femtosecond laser pulses to control the outcome of nonlinear optical process and thus to achieve the selectivity for multiphoton molecular transitions. This research could lead to applications in various fields including nonlinear optical spectroscopy, chemical identification, biological imaging, communications, photodynamic therapy, etc. In order to realize accurate pulse shaping of the femtosecond laser pulses, it is essential to measure and correct the spectral phase distortion of such pulses. A method called multiphoton intrapulse interference phase scan is used to do so throughout this dissertation. This method is highly accurate and reproducible, and has been proved in this work to be compatible with any femtosecond pulses regardless of bandwidth, intensity and repetition rate of the laser. The phase control of several quasi-octave laser sources is demonstrated in this dissertation, with the generation of 4.3 fs and 5.9 fs pulses that reach the theoretically predicted transform-limited pulse duration. The excellent phase control achieved also guarantees the reproducibility for selective multiphoton excitations by accurate phase and/or amplitude shaping. Selective two-photon excitation, stimulated Raman scattering and coherent anti-Stokes Raman scattering with a single broadband laser source are demonstrated in this dissertation. Pulse shaping is used to achieve a fast and robust approach to measure the two-photon excitation spectrum from fluorescent molecules, which provide important information for two-photon biological imaging. The selective excitation concept is also applied in the field of remote chemical identification. Detection of characteristic Raman lines for several chemicals using a single beam coherent anti-Stokes Raman scattering spectroscopy from a 12 meter standoff distance is shown, providing a promising approach to standoff detection of chemicals, hazardous contaminations

  14. Spatially resolved spectral-imaging device

    SciTech Connect

    Bloom, Joshua Simon; Tyson, John Anthony

    2016-02-09

    A spatially resolved spectral device comprising a dispersive array to receive an incident light comprising a principal ray. The dispersive array comprising a plurality of dichroic layers, each of the plurality of dichroic layers disposed in a path of a direction of the principal ray. Each of the plurality of dichroic layers configured to at least one of reflect or transmit a different wavelength range of the incident light. The device further comprising a detection array operatively coupled with the dispersive array. The detection array comprising a photosensitive component including a plurality of detection pixels, each of the plurality of detection pixels having a light-receiving surface disposed parallel to the direction of the principal ray to detect a respective one of the different wavelength ranges of incident light reflected from a corresponding one of the plurality of dichroic layers.

  15. Time-resolved spectral imaging: better photon economy, higher accuracy

    NASA Astrophysics Data System (ADS)

    Fereidouni, Farzad; Reitsma, Keimpe; Blab, Gerhard A.; Gerritsen, Hans C.

    2015-03-01

    Lifetime and spectral imaging are complementary techniques that offer a non-invasive solution for monitoring metabolic processes, identifying biochemical compounds, and characterizing their interactions in biological tissues, among other tasks. Newly developed instruments that perform time-resolved spectral imaging can provide even more information and reach higher sensitivity than either modality alone. Here we report a multispectral lifetime imaging system based on a field-programmable gate array (FPGA), capable of operating at high photon count rates (12 MHz) per spectral detection channel, and with time resolution of 200 ps. We performed error analyses to investigate the effect of gate width and spectral-channel width on the accuracy of estimated lifetimes and spectral widths. Temporal and spectral phasors were used for analysis of recorded data, and we demonstrated blind un-mixing of the fluorescent components using information from both modalities. Fractional intensities, spectra, and decay curves of components were extracted without need for prior information. We further tested this approach with fluorescently doubly-labeled DNA, and demonstrated its suitability for accurately estimating FRET efficiency in the presence of either non-interacting or interacting donor molecules.

  16. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

    PubMed Central

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.

    2016-01-01

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics. PMID:27872481

  17. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

    NASA Astrophysics Data System (ADS)

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.

    2016-11-01

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

  18. Face Recognition with Multi-Resolution Spectral Feature Images

    PubMed Central

    Sun, Zhan-Li; Lam, Kin-Man; Dong, Zhao-Yang; Wang, Han; Gao, Qing-Wei; Zheng, Chun-Hou

    2013-01-01

    The one-sample-per-person problem has become an active research topic for face recognition in recent years because of its challenges and significance for real-world applications. However, achieving relatively higher recognition accuracy is still a difficult problem due to, usually, too few training samples being available and variations of illumination and expression. To alleviate the negative effects caused by these unfavorable factors, in this paper we propose a more accurate spectral feature image-based 2DLDA (two-dimensional linear discriminant analysis) ensemble algorithm for face recognition, with one sample image per person. In our algorithm, multi-resolution spectral feature images are constructed to represent the face images; this can greatly enlarge the training set. The proposed method is inspired by our finding that, among these spectral feature images, features extracted from some orientations and scales using 2DLDA are not sensitive to variations of illumination and expression. In order to maintain the positive characteristics of these filters and to make correct category assignments, the strategy of classifier committee learning (CCL) is designed to combine the results obtained from different spectral feature images. Using the above strategies, the negative effects caused by those unfavorable factors can be alleviated efficiently in face recognition. Experimental results on the standard databases demonstrate the feasibility and efficiency of the proposed method. PMID:23418451

  19. Spectral characterization of storage phosphor imaging plates

    SciTech Connect

    Deresch, A.; Jechow, M.; Gollwitzer, C.

    2014-02-18

    This work presents attenuation and sensitivity measurements of radiographic imaging plates (IPs) with quasimonoenergetic X-rays in the 8–60 kiloelectronvolt range. The measurements are used to validate theoretical predictions. A short overview of the theoretical model is given. The model can be used to describe the sensitivity of different detector types to a wide range of X-ray energies.

  20. Detection of illicit substances in fingerprints by infrared spectral imaging.

    PubMed

    Ng, Ping Hei Ronnie; Walker, Sarah; Tahtouh, Mark; Reedy, Brian

    2009-08-01

    FTIR and Raman spectral imaging can be used to simultaneously image a latent fingerprint and detect exogenous substances deposited within it. These substances might include drugs of abuse or traces of explosives or gunshot residue. In this work, spectral searching algorithms were tested for their efficacy in finding targeted substances deposited within fingerprints. "Reverse" library searching, where a large number of possibly poor-quality spectra from a spectral image are searched against a small number of high-quality reference spectra, poses problems for common search algorithms as they are usually implemented. Out of a range of algorithms which included conventional Euclidean distance searching, the spectral angle mapper (SAM) and correlation algorithms gave the best results when used with second-derivative image and reference spectra. All methods tested gave poorer performances with first derivative and undifferentiated spectra. In a search against a caffeine reference, the SAM and correlation methods were able to correctly rank a set of 40 confirmed but poor-quality caffeine spectra at the top of a dataset which also contained 4,096 spectra from an image of an uncontaminated latent fingerprint. These methods also successfully and individually detected aspirin, diazepam and caffeine that had been deposited together in another fingerprint, and they did not indicate any of these substances as a match in a search for another substance which was known not to be present. The SAM was used to successfully locate explosive components in fingerprints deposited on silicon windows. The potential of other spectral searching algorithms used in the field of remote sensing is considered, and the applicability of the methods tested in this work to other modes of spectral imaging is discussed.

  1. Human bladder cancer diagnosis using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Mukherjee, Sushmita; Wysock, James S.; Ng, Casey K.; Akhtar, Mohammed; Perner, Sven; Lee, Ming-Ming; Rubin, Mark A.; Maxfield, Frederick R.; Webb, Watt W.; Scherr, Douglas S.

    2009-02-01

    At the time of diagnosis, approximately 75% of bladder cancers are non-muscle invasive. Appropriate diagnosis and surgical resection at this stage improves prognosis dramatically. However, these lesions, being small and/or flat, are often missed by conventional white-light cystoscopes. Furthermore, it is difficult to assess the surgical margin for negativity using conventional cystoscopes. Resultantly, the recurrence rates in patients with early bladder cancer are very high. This is currently addressed by repeat cystoscopies and biopsies, which can last throughout the life of a patient, increasing cost and patient morbidity. Multiphoton endoscopes offer a potential solution, allowing real time, noninvasive biopsies of the human bladder, as well as an up-close assessment of the resection margin. While miniaturization of the Multiphoton microscope into an endoscopic format is currently in progress, we present results here indicating that Multiphoton imaging (using a bench-top Multiphoton microscope) can indeed identify cancers in fresh, unfixed human bladder biopsies. Multiphoton images are acquired in two channels: (1) broadband autofluorescence from cells, and (2) second harmonic generation (SHG), mostly by tissue collagen. These images are then compared with gold standard hematoxylin/eosin (H&E) stained histopathology slides from the same specimen. Based on a "training set" and a very small "blinded set" of samples, we have found excellent correlation between the Multiphoton and histopathological diagnoses. A larger blinded analysis by two independent uropathologists is currently in progress. We expect that the conclusion of this phase will provide us with diagnostic accuracy estimates, as well as the degree of inter-observer heterogeneity.

  2. Image and spectral fidelity study of hyperspectral remote sensing image scaling up based on wavelet transform

    NASA Astrophysics Data System (ADS)

    An, Ni; Ma, Yi; Bao, Yuhai

    2015-08-01

    Wavelet transform is a kind of effective image-scale transformation method, which can achieve multi-scale transformation by distinguishing the low-frequency information and the high-frequency information. Hyperspectral remote sensing data combining image with spectrum has almost continuous spectrum that is the important premise of extracting hyperspectral image information, while scale transformation will inevitably lead to the change of image and spectra. Therefore, it is important to study the image and spectral fidelity after wavelet transform. In this paper, the Proba CHRIS hyperspectral remote sensing image of Yellow River Estuary Wetland is used to investigate the image and spectral fidelity of image transformed by wavelet which remained the low-frequency information. The level 1-3 of up-scale images are obtained and then compared with the original. Then image and spectral fidelity is quantitatively analyzed. The results show that the image fidelity is slightly reduced by up-scale transformation, but near-infrared images have a larger distortion than other bands. With the increasing scaling up, the distortion of spectrum is more and more great, but spectral fidelity is overall well. For the typical wetland objects, Phragmites austrialis has the best spectral correlation, Spartina has a small spectra change, and aquaculture water spectral distortion is most remarkable.

  3. High-resolution multiphoton cryomicroscopy.

    PubMed

    König, Karsten; Uchugonova, Aisada; Breunig, Hans Georg

    2014-03-15

    An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below -10 °C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20 mW mean powers.

  4. Applications of spectral imaging using a tunable laser source

    NASA Astrophysics Data System (ADS)

    Oertel, David C.; Grothaus, Jeffrey T.; Marcott, Curtis

    2009-05-01

    Hyperspectral reflectance imaging in the visible and NIR spectral ranges has considerable utility for revealing spatial and chemical complexity in both biological systems and manufactured products. Conventional imaging systems are based on broad-band illumination in tandem with a spectrometer or tunable filter placed between the sample and the detector. These systems are typically slow (require seconds of integration per wavelength step), and the CW broad-band source can cause significant heating of the sample. An alternative method is to use a tunable, pulsed, high-peak-power (low average power) source coupled with a broad-band detector. This approach offers a reduction in data acquisition time, the inherent ability to stop motion, and data collection at ambient temperature. An integrated system based on a 5- ns pulsed laser tunable from 430 nm to 2150 nm has been used to obtain hyperspectral images in both the visible and NIR spectral ranges. A number of camera/lens options allow for varied spectral bandwidths and the FOV, ranging from 11 × 15 mm2 to 15 × 20 cm2. An entire hyperspectral image stack can be collected in as little as 20 s. This method, allowing fast, room-temperature data acquisition, has sufficient sensitivity to produce data that can be successfully processed using spectral derivatives and multivariate analysis. We discuss several applications, both in vivo and otherwise, of this alternative approach to visible/NIR hyperspectral imaging.

  5. Molecular application of spectral photoacoustic imaging in pancreatic cancer pathology

    NASA Astrophysics Data System (ADS)

    Lakshman, Minalini; Hupple, Clinton; Lohse, Ines; Hedley, David; Needles, Andrew; Theodoropoulos, Catherine

    2012-12-01

    Spectral imaging is an advanced photo-acoustic (PA) mode that can discern optical absorption of contrast agent(s) in the tissue micro-environment. This advancement is made possible by precise control of optical wavelength using a tunable pulsed laser, ranging from 680-970 nm. Differential optical absorption of blood oxygenation states makes spectral imaging of hemoglobin ideal to investigate remodeling of the tumor microenvironment- a molecular change that renders resistance to standard cancer treatment. Approach: Photo-acoustic imaging was performed on the Vevo® LAZR system (VisualSonics) at 5-20 Hz. Deep abdominal imaging was accomplished with a LZ250D probe at a center frequency of 21MHz and an axial resolution of 75 μm. The tumor model was generated in an immune compromised mouse by surgical implantation of primary patient derived tumors, in the pancreas. Results: Spectral imaging for oxygen saturation at 750 nm and 850 nm characterized this tumor with a poorly oxygenated core surrounded by a well oxygenated periphery. Multispectral imaging identified a sub region in the core with a four-fold signal exclusively at 750 and 800 nm. A co-registered 2D image of this region was shown to be echogenic and calcification was suspected. Perfusion imaging with contrast enhanced ultrasound using microbubbles (Vevo MicroMarker® contrast agents, VisualSonics) identified functional vessels towards this sub region. Histology confirmed calcification and vascularization in the tumor core. Taken together, non-invasive characterization of the tumor microenvironment using photo-acoustics rendered spectral imaging a sensitive tool to monitor molecular changes representative of progression of pancreatic cancer that kills within 6 months of diagnosis.

  6. Specialized Color Targets for Spectral Reflectance Reconstruction of Magnified Images

    NASA Astrophysics Data System (ADS)

    Kruschwitz, Jennifer D. T.

    Digital images are used almost exclusively instead of film to capture visual information across many scientific fields. The colorimetric color representation within these digital images can be relayed from the digital counts produced by the camera with the use of a known color target. In image capture of magnified images, there is currently no reliable color target that can be used at multiple magnifications and give the user a solid understanding of the color ground truth within those images. The first part of this dissertation included the design, fabrication, and testing of a color target produced with optical interference coated microlenses for use in an off-axis illumination, compound microscope. An ideal target was designed to increase the color gamut for colorimetric imaging and provide the necessary "Block Dye" spectral reflectance profiles across the visible spectrum to reduce the number of color patches necessary for multiple filter imaging systems that rely on statistical models for spectral reflectance reconstruction. There are other scientific disciplines that can benefit from a specialized color target to determine the color ground truth in their magnified images and perform spectral estimation. Not every discipline has the luxury of having a multi-filter imaging system. The second part of this dissertation developed two unique ways of using an interference coated color mirror target: one that relies on multiple light-source angles, and one that leverages a dynamic color change with time. The source multi-angle technique would be used for the microelectronic discipline where the reconstructed spectral reflectance would be used to determine a dielectric film thickness on a silicon substrate, and the time varying technique would be used for a biomedical example to determine the thickness of human tear film.

  7. Surface science capabilities from IMP spectral imaging

    NASA Technical Reports Server (NTRS)

    Singer, Robert B.

    1994-01-01

    The Imager for Mars Pathfinder (IMP) had a single 12-position filter wheel for one of its two 'eyes'. Originally eight, and then nine, of these filters were optimized for surface science, and three narrow-band filters for atmospheric science. Because of some design revisions we will now have filter wheels on both sides. The wheels for right and left eyes are identical, 12 filter positions each, and rigidly linked to the same rotation shaft. There are now 13 surface filters, in addition to 5 for atmospheric observations. Details of all the filter positions are tabulated and approximate gaussian bandpasses for the 13 surface filters are shown.

  8. Fourier Spectral Filter Array for Optimal Multispectral Imaging.

    PubMed

    Jia, Jie; Barnard, Kenneth J; Hirakawa, Keigo

    2016-04-01

    Limitations to existing multispectral imaging modalities include speed, cost, range, spatial resolution, and application-specific system designs that lack versatility of the hyperspectral imaging modalities. In this paper, we propose a novel general-purpose single-shot passive multispectral imaging modality. Central to this design is a new type of spectral filter array (SFA) based not on the notion of spatially multiplexing narrowband filters, but instead aimed at enabling single-shot Fourier transform spectroscopy. We refer to this new SFA pattern as Fourier SFA, and we prove that this design solves the problem of optimally sampling the hyperspectral image data.

  9. Determination of chromophore distribution in skin by spectral imaging

    NASA Astrophysics Data System (ADS)

    Saknite, Inga; Lange, Marta; Jakovels, Dainis; Spigulis, Janis

    2012-10-01

    Possibilities to determine chromophore distribution in skin by spectral imaging were explored. Simple RGB sensor devices were used for image acquisition. Totally 200 images of 40 different bruises of 20 people were obtained in order to map chromophores bilirubin and haemoglobin. Possibilities to detect water in vitro and in vivo were estimated by using silicon photodetectors and narrow band LEDs. The results show that it is possible to obtain bilirubin and haemoglobin distribution maps and observe changes of chromophore parameter values over time by using a simple RGB imaging device. Water in vitro was detected by using differences in absorption at 450 nm and 950 nm, and 650 nm and 950 nm.

  10. Spectral image analysis of mutual illumination between florescent objects.

    PubMed

    Tominaga, Shoji; Kato, Keiji; Hirai, Keita; Horiuchi, Takahiko

    2016-08-01

    This paper proposes a method for modeling and component estimation of the spectral images of the mutual illumination phenomenon between two fluorescent objects. First, we briefly describe the bispectral characteristics of a single fluorescent object, which are summarized as a Donaldson matrix. We suppose that two fluorescent objects with different bispectral characteristics are located close together under a uniform illumination. Second, we model the mutual illumination between two objects. It is shown that the spectral composition of the mutual illumination is summarized with four components: (1) diffuse reflection, (2) diffuse-diffuse interreflection, (3) fluorescent self-luminescence, and (4) interreflection by mutual fluorescent illumination. Third, we develop algorithms for estimating the spectral image components from the observed images influenced by the mutual illumination. When the exact Donaldson matrices caused by the mutual illumination influence are unknown, we have to solve a non-linear estimation problem to estimate both the spectral functions and the location weights. An iterative algorithm is then proposed to solve the problem based on the alternate estimation of the spectral functions and the location weights. In our experiments, the feasibility of the proposed method is shown in three cases: the known Donaldson matrices, weak interreflection, and strong interreflection.

  11. Spectral prior image constrained compressed sensing (spectral PICCS) for photon-counting computed tomography

    NASA Astrophysics Data System (ADS)

    Yu, Zhicong; Leng, Shuai; Li, Zhoubo; McCollough, Cynthia H.

    2016-09-01

    Photon-counting computed tomography (PCCT) is an emerging imaging technique that enables multi-energy imaging with only a single scan acquisition. To enable multi-energy imaging, the detected photons corresponding to the full x-ray spectrum are divided into several subgroups of bin data that correspond to narrower energy windows. Consequently, noise in each energy bin increases compared to the full-spectrum data. This work proposes an iterative reconstruction algorithm for noise suppression in the narrower energy bins used in PCCT imaging. The algorithm is based on the framework of prior image constrained compressed sensing (PICCS) and is called spectral PICCS; it uses the full-spectrum image reconstructed using conventional filtered back-projection as the prior image. The spectral PICCS algorithm is implemented using a constrained optimization scheme with adaptive iterative step sizes such that only two tuning parameters are required in most cases. The algorithm was first evaluated using computer simulations, and then validated by both physical phantoms and in vivo swine studies using a research PCCT system. Results from both computer-simulation and experimental studies showed substantial image noise reduction in narrow energy bins (43-73%) without sacrificing CT number accuracy or spatial resolution.

  12. Information-Efficient Spectral Imaging Sensor With Tdi

    DOEpatents

    Rienstra, Jeffrey L.; Gentry, Stephen M.; Sweatt, William C.

    2004-01-13

    A programmable optical filter for use in multispectral and hyperspectral imaging employing variable gain time delay and integrate arrays. A telescope focuses an image of a scene onto at least one TDI array that is covered by a multispectral filter that passes separate bandwidths of light onto the rows in the TDI array. The variable gain feature of the TDI array allows individual rows of pixels to be attenuated individually. The attenuations are functions of the magnitudes of the positive and negative components of a spectral basis vector. The spectral basis vector is constructed so that its positive elements emphasize the presence of a target and its negative elements emphasize the presence of the constituents of the background of the imaged scene. This system provides for a very efficient determination of the presence of the target, as opposed to the very data intensive data manipulations that are required in conventional hyperspectral imaging systems.

  13. Spectral-Spatial Hyperspectral Image Classification Based on KNN

    NASA Astrophysics Data System (ADS)

    Huang, Kunshan; Li, Shutao; Kang, Xudong; Fang, Leyuan

    2016-12-01

    Fusion of spectral and spatial information is an effective way in improving the accuracy of hyperspectral image classification. In this paper, a novel spectral-spatial hyperspectral image classification method based on K nearest neighbor (KNN) is proposed, which consists of the following steps. First, the support vector machine is adopted to obtain the initial classification probability maps which reflect the probability that each hyperspectral pixel belongs to different classes. Then, the obtained pixel-wise probability maps are refined with the proposed KNN filtering algorithm that is based on matching and averaging nonlocal neighborhoods. The proposed method does not need sophisticated segmentation and optimization strategies while still being able to make full use of the nonlocal principle of real images by using KNN, and thus, providing competitive classification with fast computation. Experiments performed on two real hyperspectral data sets show that the classification results obtained by the proposed method are comparable to several recently proposed hyperspectral image classification methods.

  14. Content-based hyperspectral image retrieval using spectral unmixing

    NASA Astrophysics Data System (ADS)

    Plaza, Antonio J.

    2011-11-01

    The purpose of content-based image retrieval (CBIR) is to retrieve, from real data stored in a database, information that is relevant to a query. A major challenge for the development of efficient CBIR systems in the context of hyperspectral remote sensing applications is how to deal with the extremely large volumes of data produced by current Earth-observing (EO) imaging spectrometers. The data resulting from EO campaigns often comprises many Gigabytes per flight. When multiple instruments or timelines are combined, this leads to the collection of massive amounts of data coming from heterogeneous sources, and these data sets need to be effectively stored, managed, shared and retrieved. Furthermore, the growth in size and number of hyperspectral data archives demands more sophisticated search capabilities to allow users to locate and reuse data acquired in the past. In this paper we develop a new strategy to effectively retrieve hyperspectral image data sets using spectral unmixing concepts. Spectral unmixing is a very important task for hyperspectral data exploitation since the spectral signatures collected in natural environments are invariably a mixture of the pure signatures of the various materials found within the spatial extent of the ground instantaneous field view of the imaging instrument. In this work, we use the information provided by spectral unmixing (i.e. the spectral endmembers and their corresponding abundances in the scene) as effective meta-data to develop a new CBIR system that can assist users in the task of efficiently searching hyperspectral image instances in large data repositories. The proposed approach is validated using a collection of 154 hyperspectral data sets (comprising seven full flightlines) gathered by NASA using the Airborne Visible Infra-Red Imaging Spectrometer (AVIRIS) over the World Trade Center (WTC) area in New York City during the last two weeks of September, 2001, only a few days after the terrorist attacks that

  15. Differential Multiphoton Laser Scanning Microscopy

    PubMed Central

    Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

    2016-01-01

    Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot. PMID:27390511

  16. Spatial, Temporal and Spectral Satellite Image Fusion via Sparse Representation

    NASA Astrophysics Data System (ADS)

    Song, Huihui

    Remote sensing provides good measurements for monitoring and further analyzing the climate change, dynamics of ecosystem, and human activities in global or regional scales. Over the past two decades, the number of launched satellite sensors has been increasing with the development of aerospace technologies and the growing requirements on remote sensing data in a vast amount of application fields. However, a key technological challenge confronting these sensors is that they tradeoff between spatial resolution and other properties, including temporal resolution, spectral resolution, swath width, etc., due to the limitations of hardware technology and budget constraints. To increase the spatial resolution of data with other good properties, one possible cost-effective solution is to explore data integration methods that can fuse multi-resolution data from multiple sensors, thereby enhancing the application capabilities of available remote sensing data. In this thesis, we propose to fuse the spatial resolution with temporal resolution and spectral resolution, respectively, based on sparse representation theory. Taking the study case of Landsat ETM+ (with spatial resolution of 30m and temporal resolution of 16 days) and MODIS (with spatial resolution of 250m ~ 1km and daily temporal resolution) reflectance, we propose two spatial-temporal fusion methods to combine the fine spatial information of Landsat image and the daily temporal resolution of MODIS image. Motivated by that the images from these two sensors are comparable on corresponding bands, we propose to link their spatial information on available Landsat- MODIS image pair (captured on prior date) and then predict the Landsat image from the MODIS counterpart on prediction date. To well-learn the spatial details from the prior images, we use a redundant dictionary to extract the basic representation atoms for both Landsat and MODIS images based on sparse representation. Under the scenario of two prior Landsat

  17. Hyperspectral imaging of bruises in the SWIR spectral region

    NASA Astrophysics Data System (ADS)

    Randeberg, Lise L.; Hernandez-Palacios, Julio

    2012-02-01

    Optical diagnostics of bruised skin might provide important information for characterization and age determination of such injuries. Hyperspectral imaging is one of the optical techniques that have been employed for bruise characterization. This technique combines high spatial and spectral resolution and makes it possible to study both chromophore signatures and -distributions in an injury. Imaging and spectroscopy in the visible spectral range have resulted in increased knowledge about skin bruises. So far the SWIR region has not been explored for this application. The main objective of the current study was to characterize bruises in the SWIR wavelength range. Hyperspectral images in the SWIR (950-2500nm ) and VNIR (400-850nm) spectral range were collected from 3 adult volunteers with bruises of known age. Data were collected over a period of 8 days. The data were analyzed using spectroscopic techniques and statistical image analysis. Preliminary results from the pilot study indicate that SWIR hyperspectral imaging might be an important supplement to imaging in the visible part of the spectrum. The technique emphasizes local edema and gives a possibility to visualize features that cannot easily be seen in the visible part of the spectrum.

  18. Quantitative analysis of multi-spectral fundus images.

    PubMed

    Styles, I B; Calcagni, A; Claridge, E; Orihuela-Espina, F; Gibson, J M

    2006-08-01

    We have developed a new technique for extracting histological parameters from multi-spectral images of the ocular fundus. The new method uses a Monte Carlo simulation of the reflectance of the fundus to model how the spectral reflectance of the tissue varies with differing tissue histology. The model is parameterised by the concentrations of the five main absorbers found in the fundus: retinal haemoglobins, choroidal haemoglobins, choroidal melanin, RPE melanin and macular pigment. These parameters are shown to give rise to distinct variations in the tissue colouration. We use the results of the Monte Carlo simulations to construct an inverse model which maps tissue colouration onto the model parameters. This allows the concentration and distribution of the five main absorbers to be determined from suitable multi-spectral images. We propose the use of "image quotients" to allow this information to be extracted from uncalibrated image data. The filters used to acquire the images are selected to ensure a one-to-one mapping between model parameters and image quotients. To recover five model parameters uniquely, images must be acquired in six distinct spectral bands. Theoretical investigations suggest that retinal haemoglobins and macular pigment can be recovered with RMS errors of less than 10%. We present parametric maps showing the variation of these parameters across the posterior pole of the fundus. The results are in agreement with known tissue histology for normal healthy subjects. We also present an early result which suggests that, with further development, the technique could be used to successfully detect retinal haemorrhages.

  19. Retinal oxygen saturation evaluation by multi-spectral fundus imaging

    NASA Astrophysics Data System (ADS)

    Khoobehi, Bahram; Ning, Jinfeng; Puissegur, Elise; Bordeaux, Kimberly; Balasubramanian, Madhusudhanan; Beach, James

    2007-03-01

    Purpose: To develop a multi-spectral method to measure oxygen saturation of the retina in the human eye. Methods: Five Cynomolgus monkeys with normal eyes were anesthetized with intramuscular ketamine/xylazine and intravenous pentobarbital. Multi-spectral fundus imaging was performed in five monkeys with a commercial fundus camera equipped with a liquid crystal tuned filter in the illumination light path and a 16-bit digital camera. Recording parameters were controlled with software written specifically for the application. Seven images at successively longer oxygen-sensing wavelengths were recorded within 4 seconds. Individual images for each wavelength were captured in less than 100 msec of flash illumination. Slightly misaligned images of separate wavelengths due to slight eye motion were registered and corrected by translational and rotational image registration prior to analysis. Numerical values of relative oxygen saturation of retinal arteries and veins and the underlying tissue in between the artery/vein pairs were evaluated by an algorithm previously described, but which is now corrected for blood volume from averaged pixels (n > 1000). Color saturation maps were constructed by applying the algorithm at each image pixel using a Matlab script. Results: Both the numerical values of relative oxygen saturation and the saturation maps correspond to the physiological condition, that is, in a normal retina, the artery is more saturated than the tissue and the tissue is more saturated than the vein. With the multi-spectral fundus camera and proper registration of the multi-wavelength images, we were able to determine oxygen saturation in the primate retinal structures on a tolerable time scale which is applicable to human subjects. Conclusions: Seven wavelength multi-spectral imagery can be used to measure oxygen saturation in retinal artery, vein, and tissue (microcirculation). This technique is safe and can be used to monitor oxygen uptake in humans. This work

  20. Mesospheric nightglow spectral survey taken by the ISO spectral spatial imager on Atlas 1

    NASA Technical Reports Server (NTRS)

    Owens, J. K.; Torr, D. G.; Torr, M. R.; Chang, T.; Fennelly, J. A.; Richards, P. G.; Morgan, M. F.; Baldridge, T. W.; Dougani, H.; Swift, W.

    1993-01-01

    This paper reports the first comprehensive spectral survey of the mesospheric airglow between 260 and 832 nm taken by the Imaging Spectrometric Observatory (ISO) on the ATLAS I mission. We select data taken in the spectral window between 275 and 300 nm to determine the variation with altitude of the Herzberg I bands originating from the vibrational levels v' = 3 to 8. These data provide the first spatially resolved spectral measurements of the system. The data are used to demonstrate that to within an uncertainty of + 10%, the vibrational distribution remains invariant with altitude. The deficit reported previously for the v' = 5 level is not observed although there is a suggestion of depletion in v' = 6. The data could be used to place tight constraints on the vibrational dependence of quenching rate coefficients, and on the abundance of atomic oxygen.

  1. Mesospheric nightglow spectral survey taken by the ISO spectral spatial imager on ATLAS 1

    NASA Technical Reports Server (NTRS)

    Owens, J. K.; Torr, D. G.; Torr, M. R.; Chang, T.; Fennelly, J. A.; Richards, P. G.; Morgan, M. F.; Baldridge, T. W.; Fellows, C. W.; Dougani, H.

    1993-01-01

    This paper reports the first comprehensive spectral survey of the mesospheric airglow between 260 and 832 nm taken by the Imaging Spectrometric Observatory on the ATLAS 1 mission. We select data taken in the spectral window between 275 and 300 nm to determine the variation with altitude of the Herzberg I bands originating from the vibrational levels v-prime = 3 to 8. These data provide the first spatially resolved spectral measurements of the system. The data are used to demonstrate that to within an uncertainty of +/- 10 percent, the vibrational distribution remains invariant with altitude. The deficit reported previously for the v-prime = 5 level is not observed although there is a suggestion of depletion in v-prime = 6. The data could be used to place tight constraints on the vibrational dependence of quenching rate coefficients, and on the abundance of atomic oxygen.

  2. Clinical multiphoton endoscopy with FLIM capability

    NASA Astrophysics Data System (ADS)

    Weinigel, Martin; Breunig, Hans Georg; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; König, Karsten

    2013-02-01

    Multiphoton endoscopy can be applied for intra-corporeal imaging as well as to examine otherwise hard-to-access tissue areas like chronic wounds. Using high-NA (NA = 0.8) gradient-index (GRIN) lens-based endoscopes with a diameter of 1.4 mm and effective lengths of 7 mm and 20 mm, respectively, two-photon excitation of endogenous fluorophores and second-harmonic generation (SHG) is used for multimodal in vivo imaging of human skin. A further imaging modality is fluorescence lifetime imaging (FLIM) which allows functional imaging to investigate the healing mechanism of chronic wounds and the corresponding cell metabolism. We performed first in vivo measurements using FLIM endoscopy with the medically-certified multiphoton tomograph MPTflex® in combination with a computer-controlled motorized scan head and a GRIN-lens endoscope.

  3. Rapid microscopy measurement of very large spectral images.

    PubMed

    Lindner, Moshe; Shotan, Zav; Garini, Yuval

    2016-05-02

    The spectral content of a sample provides important information that cannot be detected by the human eye or by using an ordinary RGB camera. The spectrum is typically a fingerprint of the chemical compound, its environmental conditions, phase and geometry. Thus measuring the spectrum at each point of a sample is important for a large range of applications from art preservation through forensics to pathological analysis of a tissue section. To date, however, there is no system that can measure the spectral image of a large sample in a reasonable time. Here we present a novel method for scanning very large spectral images of microscopy samples even if they cannot be viewed in a single field of view of the camera. The system is based on capturing information while the sample is being scanned continuously 'on the fly'. Spectral separation implements Fourier spectroscopy by using an interferometer mounted along the optical axis. High spectral resolution of ~5 nm at 500 nm could be achieved with a diffraction-limited spatial resolution. The acquisition time is fairly high and takes 6-8 minutes for a sample size of 10mm x 10mm measured under a bright-field microscope using a 20X magnification.

  4. Simulating Colour Vision Deficiency from a Spectral Image.

    PubMed

    Shrestha, Raju

    2016-01-01

    People with colour vision deficiency (CVD) have difficulty seeing full colour contrast and can miss some of the features in a scene. As a part of universal design, researcher have been working on how to modify and enhance the colour of images in order to make them see the scene with good contrast. For this, it is important to know how the original colour image is seen by different individuals with CVD. This paper proposes a methodology to simulate accurate colour deficient images from a spectral image using cone sensitivity of different cases of deficiency. As the method enables generation of accurate colour deficient image, the methodology is believed to help better understand the limitations of colour vision deficiency and that in turn leads to the design and development of more effective imaging technologies for better and wider accessibility in the context of universal design.

  5. Spectral imaging and biomedicine: new devices, new approaches

    SciTech Connect

    Levenson, Richard M.; Cronin, P. J.; Harvey, N. R.

    2002-01-01

    The advent of molecular medicine and new demands on pathologists to deliver prognostic and therapy-shaping analyses has created a need for enhanced imaging tools. Spectral imaging coupled wilh microscopy is a relatively novel and largely unexplored technology that holds out promise of satisfying, at least in part, such a need. New optical methods for spectral discrimination are being combined with powerful software approaches, often originally developed in diffbrent fields, to explore and exploit a wealth of informaticm beyond the capabilities of conventional color-based imaging approaches. Some of the new devices and software tools are described and illustrated here. While the results are indeed promising, it must be stressed that this field is in its infancy, and the optimal uses of this technology in the clinical arena still await definition.

  6. Acousto-optic infrared spectral imager for Pluto fast flyby

    NASA Technical Reports Server (NTRS)

    Glenar, D. A.; Hillman, J. J.

    1993-01-01

    Acousto-optic tunable filters (AOTF's) enable the design of compact, two-dimensional imaging spectrometers with high spectral and spatial resolution and with no moving parts. Tellurium dioxide AOTF's operate from about 400 nm to nearly 5 microns, and a single device will tune continuously over one octave by changing the RF acoustic frequency applied to the device. An infrared (1.2-2.5 micron) Acousto-Optic Imaging Spectrometer (AImS) was designed that closely conforms to the surface composition mapping objectives of the Pluto Fast Flyby. It features a 75-cm focal length telescope, infrared AOTF, and 256 x 256 NICMOS-3 focal plane array for acquiring narrowband images with a spectral resolving power (lambda/delta(lambda)) exceeding 250. We summarize the instrument design features and its expected performance at the Pluto-Charon encounter.

  7. Differential Multiphoton Laser Scanning Microscopy

    SciTech Connect

    Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

    2012-01-01

    Multifocal multiphoton laser scanning microscopy (mfMPLSM) in the biological and medical sciences has the potential to become a ubiquitous tool for obtaining high-resolution images at video rates. While current implementations of mfMPLSM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for mfMPLSM in which whole-field detection with a single detector, rather than detection with a matrix of detectors, such as a charge-coupled device (CCD) camera, is implemented. This advance makes mfMPLSM fully compatible for use in imaging through scattering media. Further, we demonstrate that this method makes it possible to simultaneously obtain multiple images and view differences in excitation parameters in a single scan of the specimen.

  8. Hindlimb heating increases vascular access of large molecules to murine tibial growth plates measured by in vivo multiphoton imaging.

    PubMed

    Serrat, Maria A; Efaw, Morgan L; Williams, Rebecca M

    2014-02-15

    Advances in understanding the molecular regulation of longitudinal growth have led to development of novel drug therapies for growth plate disorders. Despite progress, a major unmet challenge is delivering therapeutic agents to avascular-cartilage plates. Dense extracellular matrix and lack of penetrating blood vessels create a semipermeable "barrier," which hinders molecular transport at the vascular-cartilage interface. To overcome this obstacle, we used a hindlimb heating model to manipulate bone circulation in 5-wk-old female mice (n = 22). Temperatures represented a physiological range of normal human knee joints. We used in vivo multiphoton microscopy to quantify temperature-enhanced delivery of large molecules into tibial growth plates. We tested the hypothesis that increasing hindlimb temperature from 22°C to 34°C increases vascular access of large systemic molecules, modeled using 10, 40, and 70 kDa dextrans that approximate sizes of physiological regulators. Vascular access was quantified by vessel diameter, velocity, and dextran leakage from subperichondrial plexus vessels and accumulation in growth plate cartilage. Growth plate entry of 10 kDa dextrans increased >150% at 34°C. Entry of 40 and 70 kDa dextrans increased <50%, suggesting a size-dependent temperature enhancement. Total dextran levels in the plexus increased at 34°C, but relative leakage out of vessels was not temperature dependent. Blood velocity and vessel diameter increased 118% and 31%, respectively, at 34°C. These results demonstrate that heat enhances vascular carrying capacity and bioavailability of large molecules around growth plates, suggesting that temperature could be a noninvasive strategy for modulating delivery of therapeutics to impaired growth plates of children.

  9. Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung

    NASA Astrophysics Data System (ADS)

    Pavlova, Ina; Hume, Kelly R.; Yazinski, Stephanie A.; Flanders, James; Southard, Teresa L.; Weiss, Robert S.; Webb, Watt W.

    2012-03-01

    Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung. Ex vivo MPM imaging of intrinsic two-photon excited fluorescence was performed on mouse and canine neoplastic, inflammatory and tumor-free lung sites. Results showed that MPM detected microanatomical differences between tumor-free and neoplastic lung tissue similar to standard histopathology but without the need for tissue processing. Furthermore, inflammatory sites displayed a distinct red-shifted fluorescence compared to neoplasms in both mouse and canine lung, and adenocarcinomas displayed a less pronounced fluorescence emission in the 500 to 550 nm region compared to adenomas in mouse models of lung cancer. These spectral distinctions were also confirmed by two-photon excited fluorescence microspectroscopy. We demonstrate the feasibility of applying MPM imaging of intrinsic fluorescence for the differentiation of lung neoplasms, inflammatory and tumor-free lung, which motivates the application of multiphoton endoscopy for the in situ imaging of lung nodules.

  10. Quantum Dot-Based Hybrid Nanostructures and Energy Transfer on the Nanoscale for Single- and Multi-Photon Imaging and Cancer Diagnostics

    NASA Astrophysics Data System (ADS)

    Nabiev, Igor

    2017-01-01

    An ideal single-photon (1P) or multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible 1P or two-photon (2P) absorption cross section and the smallest possible highly specific recognition molecules conjugated with the nanoparticle in an oriented manner. However, the conditions used for conjugation of typical recognition molecules (conventional antibodies, Abs) with nanoparticles often provoke their unfolding and/or yield nanoprobes with irregular orientation of Abs on the nanoparticle surface. Conjugation of smaller Ab fragments, such as single-domain antibodies (sdAbs), with quantum dots (QDs) in an oriented manner can be considered as an attractive approach to engineering of ultrasmall diagnostic nanoprobes. QDs conjugated to 13-kDa sdAbs derived from camelid IgG or streptavidin have been used as efficient 1P or 2P excitation probes for imaging of cancer markers. The 2P absorption cross sections (TPACSs) for some conjugates are higher than 49,000 GM (Goeppert–Mayer units), which is close to the theoretical value calculated for CdSe QDs and considerably exceeds that of organic dyes. A further step in advanced QD-based cancer diagnostics has been made through implementation of efficient FRET-based imaging with 2P excitation, which has been demonstrated for double immunostaining complexes formed on the surface of cancer cells from sdAb–QD conjugates (donor) and a combination of monoclonal Abs and secondary antibodies labeled with the AlexaFluor dye (acceptor). The proposed approach permits obtaining an exceptional contrast of 2P imaging of cancer biomarkers without any contribution of cell and tissue autofluorescence in the recorded images.

  11. Spectral analysis of mammographic images using a multitaper method

    SciTech Connect

    Wu Gang; Mainprize, James G.; Yaffe, Martin J.

    2012-02-15

    Purpose: Power spectral analysis in radiographic images is conventionally performed using a windowed overlapping averaging periodogram. This study describes an alternative approach using a multitaper technique and compares its performance with that of the standard method. This tool will be valuable in power spectrum estimation of images, whose content deviates significantly from uniform white noise. The performance of the multitaper approach will be evaluated in terms of spectral stability, variance reduction, bias, and frequency precision. The ultimate goal is the development of a useful tool for image quality assurance. Methods: A multitaper approach uses successive data windows of increasing order. This mitigates spectral leakage allowing one to calculate a reduced-variance power spectrum. The multitaper approach will be compared with the conventional power spectrum method in several typical situations, including the noise power spectra (NPS) measurements of simulated projection images of a uniform phantom, NPS measurement of real detector images of a uniform phantom for two clinical digital mammography systems, and the estimation of the anatomic noise in mammographic images (simulated images and clinical mammograms). Results: Examination of spectrum variance versus frequency resolution and bias indicates that the multitaper approach is superior to the conventional single taper methods in the prevention of spectrum leakage and variance reduction. More than four times finer frequency precision can be achieved with equivalent or less variance and bias. Conclusions: Without any shortening of the image data length, the bias is smaller and the frequency resolution is higher with the multitaper method, and the need to compromise in the choice of regions of interest size to balance between the reduction of variance and the loss of frequency resolution is largely eliminated.

  12. Multi-material decomposition of spectral CT images

    NASA Astrophysics Data System (ADS)

    Mendonça, Paulo R. S.; Bhotika, Rahul; Maddah, Mahnaz; Thomsen, Brian; Dutta, Sandeep; Licato, Paul E.; Joshi, Mukta C.

    2010-04-01

    Spectral Computed Tomography (Spectral CT), and in particular fast kVp switching dual-energy computed tomography, is an imaging modality that extends the capabilities of conventional computed tomography (CT). Spectral CT enables the estimation of the full linear attenuation curve of the imaged subject at each voxel in the CT volume, instead of a scalar image in Hounsfield units. Because the space of linear attenuation curves in the energy ranges of medical applications can be accurately described through a two-dimensional manifold, this decomposition procedure would be, in principle, limited to two materials. This paper describes an algorithm that overcomes this limitation, allowing for the estimation of N-tuples of material-decomposed images. The algorithm works by assuming that the mixing of substances and tissue types in the human body has the physicochemical properties of an ideal solution, which yields a model for the density of the imaged material mix. Under this model the mass attenuation curve of each voxel in the image can be estimated, immediately resulting in a material-decomposed image triplet. Decomposition into an arbitrary number of pre-selected materials can be achieved by automatically selecting adequate triplets from an application-specific material library. The decomposition is expressed in terms of the volume fractions of each constituent material in the mix; this provides for a straightforward, physically meaningful interpretation of the data. One important application of this technique is in the digital removal of contrast agent from a dual-energy exam, producing a virtual nonenhanced image, as well as in the quantification of the concentration of contrast observed in a targeted region, thus providing an accurate measure of tissue perfusion.

  13. Online Unmixing of Multitemporal Hyperspectral Images Accounting for Spectral Variability.

    PubMed

    Thouvenin, Pierre-Antoine; Dobigeon, Nicolas; Tourneret, Jean-Yves

    2016-09-01

    Hyperspectral unmixing is aimed at identifying the reference spectral signatures composing a hyperspectral image and their relative abundance fractions in each pixel. In practice, the identified signatures may vary spectrally from an image to another due to varying acquisition conditions, thus inducing possibly significant estimation errors. Against this background, the hyperspectral unmixing of several images acquired over the same area is of considerable interest. Indeed, such an analysis enables the endmembers of the scene to be tracked and the corresponding endmember variability to be characterized. Sequential endmember estimation from a set of hyperspectral images is expected to provide improved performance when compared with methods analyzing the images independently. However, the significant size of the hyperspectral data precludes the use of batch procedures to jointly estimate the mixture parameters of a sequence of hyperspectral images. Provided that each elementary component is present in at least one image of the sequence, we propose to perform an online hyperspectral unmixing accounting for temporal endmember variability. The online hyperspectral unmixing is formulated as a two-stage stochastic program, which can be solved using a stochastic approximation. The performance of the proposed method is evaluated on synthetic and real data. Finally, a comparison with independent unmixing algorithms illustrates the interest of the proposed strategy.

  14. Tunable acousto-optic spectral imager for atmospheric composition measurements in the visible spectral domain.

    PubMed

    Dekemper, Emmanuel; Loodts, Nicolas; Van Opstal, Bert; Maes, Jeroen; Vanhellemont, Filip; Mateshvili, Nina; Franssens, Ghislain; Pieroux, Didier; Bingen, Christine; Robert, Charles; De Vos, Lieve; Aballea, Ludovic; Fussen, Didier

    2012-09-01

    We describe a new spectral imaging instrument using a TeO(2) acousto-optical tunable filter (AOTF) operating in the visible domain (450-900 nm). It allows for fast (~1 second), monochromatic (FWHM ranges from 0.6 nm at 450 nm to 3.5 nm at 800 nm) picture acquisition with good spatial resolution. This instrument was designed as a breadboard of the visible channel of a new satellite-borne atmospheric limb spectral imager, named the Atmospheric Limb Tracker for the Investigation of the Upcoming Stratosphere (ALTIUS), that is currently being developed. We tested its remote sensing capabilities by observing the dense, turbulent plume exhausted by a waste incinerator stack at two wavelengths sensitive to NO(2). An average value of 6.0±0.4×10(17) molecules cm(-2) has been obtained for the NO(2) slant column density within the plume, close to the stack outlet. Although this result was obtained with a rather low accuracy, it demonstrates the potential of spectral imaging by using AOTFs in remote sensing.

  15. Quantitative study on appearance of microvessels in spectral endoscopic imaging

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Hiroshi; Saito, Takaaki; Shiraishi, Yasushi; Arai, Fumihito; Morimoto, Yoshinori; Yuasa, Atsuko

    2015-03-01

    Increase in abnormal microvessels in the superficial mucosa is often relevant to diagnostic findings of neoplasia in digestive endoscopy; hence, observation of superficial vasculature is crucial for cancer diagnosis. To enhance the appearance of such vessels, several spectral endoscopic imaging techniques have been developed, such as narrow-band imaging and blue laser imaging. Both techniques exploit narrow-band blue light for the enhancement. The emergence of such spectral imaging techniques has increased the importance of understanding the relation of the light wavelength to the appearance of superficial vasculature, and thus a new method is desired for quantitative analysis of vessel visibility in relation to the actual structure in the tissue. Here, we developed microvessel-simulating phantoms that allowed quantitative evaluation of the appearance of 15-μm-thick vessels. We investigated the relation between the vascular contrast and light wavelength by the phantom measurements and also verified it in experiments with swine, where the endoscopically observed vascular contrast was investigated together with its real vascular depth and diameter obtained by microscopic observation of fluorescence-labeled vessels. Our study indicates that changing the spectral property even in the wavelength range of blue light may allow selective enhancement of the vascular depth for clinical use.

  16. Image features of spectral correlation function for arrhythmia classification.

    PubMed

    Khalaf, Aya F; Owis, Mohammed I; Yassine, Inas A

    2015-01-01

    Recently, computerized arrhythmia classification tools have been intensively used to aid physicians to recognize different irregular heartbeats. In this paper, we introduce arrhythmia CAD system exploiting cyclostationary signal analysis through estimation of the spectral correlation function for 5 different beat types. Two experiments were performed. Raw spectral correlation data were used as features in the first experiment while the other experiment which dealt with the spectral correlation coefficients as image included extraction of wavelet and shape features followed by fisher score for dimensionality reduction. As for the classification task, Support Vector Machine (SVM) with linear kernel was used for both experiments. The experimental results showed that both proposed approaches are superior compared to several state of the art methods. This approach achieved sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of 99.20%, 99.70%, 98.60%, 99.90% and 97.60% respectively.

  17. Comparative noise performance of a coded aperture spectral imager

    NASA Astrophysics Data System (ADS)

    Piper, Jonathan; Yuen, Peter; Godfree, Peter; Ding, Mengjia; Soori, Umair; Selvagumar, Senthurran; James, David

    2016-10-01

    Novel types of spectral sensors using coded apertures may offer various advantages over conventional designs, especially the possibility of compressive measurements that could exceed the expected spatial, temporal or spectral resolution of the system. However, the nature of the measurement process imposes certain limitations, especially on the noise performance of the sensor. This paper considers a particular type of coded-aperture spectral imager and uses analytical and numerical modelling to compare its expected noise performance with conventional hyperspectral sensors. It is shown that conventional sensors may have an advantage in conditions where signal levels are high, such as bright light or slow scanning, but that coded-aperture sensors may be advantageous in low-signal conditions.

  18. Infrared and visible image fusion with spectral graph wavelet transform.

    PubMed

    Yan, Xiang; Qin, Hanlin; Li, Jia; Zhou, Huixin; Zong, Jing-guo

    2015-09-01

    Infrared and visible image fusion technique is a popular topic in image analysis because it can integrate complementary information and obtain reliable and accurate description of scenes. Multiscale transform theory as a signal representation method is widely used in image fusion. In this paper, a novel infrared and visible image fusion method is proposed based on spectral graph wavelet transform (SGWT) and bilateral filter. The main novelty of this study is that SGWT is used for image fusion. On the one hand, source images are decomposed by SGWT in its transform domain. The proposed approach not only effectively preserves the details of different source images, but also excellently represents the irregular areas of the source images. On the other hand, a novel weighted average method based on bilateral filter is proposed to fuse low- and high-frequency subbands by taking advantage of spatial consistency of natural images. Experimental results demonstrate that the proposed method outperforms seven recently proposed image fusion methods in terms of both visual effect and objective evaluation metrics.

  19. In vivo non-invasive multiphoton tomography of human skin

    NASA Astrophysics Data System (ADS)

    König, Karsten; Riemann, Iris; Ehlers, Alexander; Le Harzic, Ronan

    2005-10-01

    High resolution non-invasive 3D imaging devices are required to detect pathogenic microorganisms such as Anthrax spores, bacteria, viruses, fungi and chemical agents entering biological tissues such as the epidermis. Due to the low light penetration depth and the biodamage potential, ultraviolet light sources can not be employed to realize intratissue imaging of bio- and chemohazards. We report on the novel near infrared laser technology multiphoton tomography and the high resolution 4D imaging tool DermaInspect for non-invasive detection of intratissue agents and their influence on cellular metabolism based on multiphoton autofluorescence imaging (MAI) and second harmonic generation (SHG). Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence of both, skin tissues and microorganisms, originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Bacteria emit in the blue/green spectral range due to NAD(P)H and flavoproteins and, in certain cases, in the red spectral range due to the biosynthesis of Zn-porphyrins, coproporphyrin and protoporphyrin. Collagen and exogenous non-centrosymmetric molecules can be detected by SHG signals. The system DermaInspect consists of a wavelength-tunable compact 80/90 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezo-driven objective, fast photon detector and time-resolved single photon counting unit. It can be used to perform optical sectioning and 3D autofluorescence lifetime imaging (τ-mapping) with 1 μm spatial resolution and 270 ps temporal resolution. The parameter fluorescence lifetime depends on the type of fluorophore and its microenvironment and can be used to distinguish bio- and chemohazards from cellular background and to gain information for pathogen

  20. LED-based endoscopic light source for spectral imaging

    NASA Astrophysics Data System (ADS)

    Browning, Craig M.; Mayes, Samuel; Favreau, Peter; Rich, Thomas C.; Leavesley, Silas J.

    2016-03-01

    Colorectal cancer is the United States 3rd leading cancer in death rates.1 The current screening for colorectal cancer is an endoscopic procedure using white light endoscopy (WLE). There are multiple new methods testing to replace WLE, for example narrow band imaging and autofluorescence imaging.2 However, these methods do not meet the need for a higher specificity or sensitivity. The goal for this project is to modify the presently used endoscope light source to house 16 narrow wavelength LEDs for spectral imaging in real time while increasing sensitivity and specificity. The process to do such was to take an Olympus CLK-4 light source, replace the light and electronics with 16 LEDs and new circuitry. This allows control of the power and intensity of the LEDs. This required a larger enclosure to house a bracket system for the solid light guide (lightpipe), three new circuit boards, a power source and National Instruments hardware/software for computer control. The results were a successfully designed retrofit with all the new features. The LED testing resulted in the ability to control each wavelength's intensity. The measured intensity over the voltage range will provide the information needed to couple the camera for imaging. Overall the project was successful; the modifications to the light source added the controllable LEDs. This brings the research one step closer to the main goal of spectral imaging for early detection of colorectal cancer. Future goals will be to connect the camera and test the imaging process.

  1. Images and Spectral Performance of WFC3 Interference Filters

    NASA Technical Reports Server (NTRS)

    Quijada, Manuel A.; Boucarut, R.; Telfer, R.; Baggett, S.; Quijano, J. Kim; Allen, George; Arsenovic, Peter

    2006-01-01

    The Wide Field Camera 3 (WFC3) is a panchromatic imager that will be deployed in the Hubble Space Telescope (HST). The mission of the WFC3 is to enhance HST1s imaging capability in the ultraviolet, visible and near-infrared spectral regions. Together with a wavelength coverage spanning 2000A to 1.7 micron, the WFC3 high sensitivity, high spatial resolution, and large field-of-view provide the astronomer with an unprecedented set of tools for exploring all types of exciting astrophysical terrain and for addressing many key questions in astronomy today. The filter compliment, which includes broad, medium, and narrow band filters, naturally reflects the diversity of astronomical programs to be targeted with WFC3. The WFC3 holds 61 UVIS filters elements, 14 IR filters, and 3 dispersive elements. During ground testing, the majority of the UVIS filters were found to exhibit excellent performance consistent with or exceeding expectations; however, a subset of filters showed considerable ghost images; some with relative intensity as high as 10-15%. Replacement filters with band-defining coatings that substantially reduce these ghost images were designed and procured. A state-of-the-art characterization setup was developed to measured the intensity of ghost images, focal shift, wedge direction , transmitted uniformity and surface feature of filters that could effect uniform flat field images. We will report on this new filter characterization methods, as well as the spectral performance measurements of the in-band transmittance and blocking.

  2. Multiphoton spectroscopy of human skin in vivo

    NASA Astrophysics Data System (ADS)

    Breunig, Hans G.; Weinigel, Martin; König, Karsten

    2012-03-01

    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.

  3. Sparse representation-based spectral clustering for SAR image segmentation

    NASA Astrophysics Data System (ADS)

    Zhang, Xiangrong; Wei, Zhengli; Feng, Jie; Jiao, Licheng

    2011-12-01

    A new method, sparse representation based spectral clustering (SC) with Nyström method, is proposed for synthetic aperture radar (SAR) image segmentation. Different from the conventional SC, this proposed technique is developed by using the sparse coefficients which obtained by solving l1 minimization problem to construct the affinity matrix and the Nyström method is applied to alleviate the segmentation process. The advantage of our proposed method is that we do not need to select the scaling parameter in the Gaussian kernel function artificially. We apply the proposed method, k-means and the classic spectral clustering algorithm with Nyström method to SAR image segmentation. The results show that compared with the other two methods, the proposed method can obtain much better segmentation results.

  4. Hybrid Image Fusion for Sharpness Enhancement of Multi-Spectral Lunar Images

    NASA Astrophysics Data System (ADS)

    Awumah, Anna; Mahanti, Prasun; Robinson, Mark

    2016-10-01

    Image fusion enhances the sharpness of a multi-spectral (MS) image by incorporating spatial details from a higher-resolution panchromatic (Pan) image [1,2]. Known applications of image fusion for planetary images are rare, although image fusion is well-known for its applications to Earth-based remote sensing. In a recent work [3], six different image fusion algorithms were implemented and their performances were verified with images from the Lunar Reconnaissance Orbiter (LRO) Camera. The image fusion procedure obtained a high-resolution multi-spectral (HRMS) product from the LRO Narrow Angle Camera (used as Pan) and LRO Wide Angle Camera (used as MS) images. The results showed that the Intensity-Hue-Saturation (IHS) algorithm results in a high-spatial quality product while the Wavelet-based image fusion algorithm best preserves spectral quality among all the algorithms. In this work we show the results of a hybrid IHS-Wavelet image fusion algorithm when applied to LROC MS images. The hybrid method provides the best HRMS product - both in terms of spatial resolution and preservation of spectral details. Results from hybrid image fusion can enable new science and increase the science return from existing LROC images.[1] Pohl, Cle, and John L. Van Genderen. "Review article multisensor image fusion in remote sensing: concepts, methods and applications." International journal of remote sensing 19.5 (1998): 823-854.[2] Zhang, Yun. "Understanding image fusion." Photogramm. Eng. Remote Sens 70.6 (2004): 657-661.[3] Mahanti, Prasun et al. "Enhancement of spatial resolution of the LROC Wide Angle Camera images." Archives, XXIII ISPRS Congress Archives (2016).

  5. Spectral Image Processing and Analysis of the Archimedes Palimpsest

    DTIC Science & Technology

    2011-09-01

    SPECTRAL IMAGE PROCESSING AND ANALYSIS OF THE ARCHIMEDES PALIMPSEST Roger L. Easton, Jr., William A. Christens-Barry, Keith T. Knox Chester F...5988 (fax), e-mail: easton@cis.rit.edu web: www.cis.rit.edu/people/faculty/easton ABSTRACT The Archimedes Palimpsest is a 10th-century parchment...rendering. 1. SIGNIFICANCE OF THE CODEX Almost everything known about the work of Archimedes has been gleaned from three codex manuscripts. The first

  6. Spectral Imaging for Revealing and Preserving World Cultural Heritage

    DTIC Science & Technology

    2011-09-01

    of appearance of the original woodcut features Other signal processing included a PCA spectral separation of an iron gall ink inscription most...likely added by Schöner, 1451 as shown in Figure 3. The image processing clearly illus- trates the iron gall ink writing on top of the printing ink ...Figure 3 – PCA of printing and iron gall ink inscription (sheet 10) Other obscured historical information included revealing the text of a

  7. Acquisition and analysis of spectral image data by linear un-mixing, cluster computing and a novel spectral imager

    NASA Astrophysics Data System (ADS)

    Barber, Paul R.; Edens, Richard J.; Vojnovic, B.

    2005-03-01

    We describe how spectral imaging, linear un-mixing and cluster computing have been combined to aid biomedical researchers and allow the spatial segmentation and quantitative analysis of immunohistochemically stained tissue section images. A novel cost-effective spectral imager, with a bandwidth of 15 nm between 400 and 700 nm, allows us to record both spatial and spectral data from absorptive and fluorescent chemical probes. The linear un-mixing of this data separates the stain distributions revealing areas of co-localisation and extracts quantitative values of optical density. This has been achieved at the single-pixel level of an image by non-negative least squares fitting. This process can be computationally expensive but great processing speed increases have been achieved through the use of cluster computing. We describe how several personal computers, running Microsoft WindowsXP, can be used in parallel, linked by the MPI (Message Passing Interface) standard. We describe how the free MPICH libraries have been incorporated into our spectral imaging application under the C language and how this has been extended to support features of MPI2 via the commercial WMPI II libraries. A cluster of 8 processors, in 4 dual-Athlon-2600+ computers, offered a speed up of a factor of 5 compared to a singleton. This includes the time required to transfer the data throughout the cluster and reflects a processing efficiency of 0.62 (a Cluster Efficacy of 3.0). The cluster was based on a 1000Base-T Ethernet network and appears to be scalable efficiently beyond 8 processors.

  8. Evaluation of the oxidative stress of psoriatic fibroblasts based on spectral two-photon fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Kapsokalyvas, Dimitrios; Barygina, Victoria; Cicchi, Riccardo; Fiorillo, Claudia; Pavone, Francesco S.

    2013-02-01

    Psoriasis is an autoimmune disease of the skin characterized by hyperkeratosis, hyperproliferation of the epidermis, inflammatory cell accumulation and increased dilatation of dermal papillary blood vessels. Metabolic activity is increased in the epidermis and the dermis. Oxidative stress is high mainly due to reactive oxygen species (ROS) originating from the skin environment and cellular metabolism. We employed a custom multiphoton microscope coupled with a FLIM setup to image primary culture fibroblast cells from perilesional and lesional psoriatic skin in-vitro. Twophoton excited fluorescence images revealed the morphological differences between healthy and psoriatic fibroblasts. Based on the spectral analysis of the NADH and FAD components the oxidative stress was assessed and found to be higher in psoriatic cells. Furthermore the fluorescence lifetime properties were investigated with a TCSPC FLIM module. Mean fluorescence lifetime was found to be longer in psoriatic lesional cells. Analysis of the fast (τ1) and slow (τ2) decay lifetimes revealed a decrease of the ratio of the contribution of the fast (α1) parameter to the contribution of the slow (α2) parameter. The fluorescence in the examined part of the spectrum is attributed mainly to NADH. The decrease of the ratio (α1)/ (α2) is believed to correlate strongly with the anti-oxidant properties of NADH which can lead to the variation of its population in high ROS environment. This methodology could serve as an index of the oxidative status in cells and furthermore could be used to probe the oxidative stress of tissues in-vivo.

  9. Noninvasive dosimetry and monitoring of TTT using spectral imaging

    NASA Astrophysics Data System (ADS)

    Schuele, G.; Molnar, F. E.; Yellachich, D.; Vitkin, E.; Perelman, L. T.; Palanker, D.

    2006-02-01

    Transpupillary thermo therapy (TTT) is a slow (60 seconds) photothermal treatment of the fundus with a near-infrared (780-810nm) laser irradiating a large spot (0.5- 1. mm) on the retina. Due to high variability in ocular tissue properties and the lack of immediately observable outcome of the therapy, a real-time dosimetry is highly desirable. We found that fundus spectroscopy and spectrally-resolved imaging allow for non-invasive real-time monitoring and dosimetry of TTT. A 795nm laser was applied in rabbit eyes for 60 seconds using a 0.86mm retinal spot diameter. The fundus was illuminated with a broadband polarized light, and its reflectance spectra were measured in parallel and cross-polarizations. The fundus was also imaged in selected spectral domains. At irradiances that do not create ophthalmoscopically visible lesions the fundus reflectance increases at the wavelengths corresponding to absorption of the oxygenated blood indicating the reduced concentration of blood in the choroid. Vasoconstrictive response of the choroidal and retinal vasculature during TTT was also directly observed using spectrally-resolved imaging. At irradiances that produce ophthalmoscopically visible lesions a rapid reduction of the fundus reflectance was observed within the first 5-10 seconds of the exposure even when the visible lesions developed only by the end of the 60 second exposure. No visible lesions were produced where the laser was terminated after detection of the reduced scattering but prior to appearance of the enhanced scattering.

  10. Solar abundances with the SPICE spectral imager on Solar Orbiter

    NASA Astrophysics Data System (ADS)

    Giunta, Alessandra; Haberreiter, Margit; Peter, Hardi; Vial, Jean-Claude; Harrison, Richard; Parenti, Susanna; Innes, Davina; Schmutz, Werner; Buchlin, Eric; Chamberlin, Phillip; Thompson, William; Bocchialini, Karine; Gabriel, Alan; Morris, Nigel; Caldwell, Martin; Auchere, Frederic; Curdt, Werner; Teriaca, Luca; Hassler, Donald M.; DeForest, Craig; Hansteen, Viggo; Carlsson, Mats; Philippon, Anne; Janvier, Miho; Wimmer-Schweingruber, Robert; Griffin, Douglas; Baudin, Frederic; Davila, Joseph; Fludra, Andrzej; Waltham, Nick; Eccleston, Paul; Gottwald, Alexander; Klein, Roman; Hanley, John; Walls, Buddy; Howe, Chris; Schuehle, Udo; Gyo, Manfred; Pfiffner, Dany

    2016-07-01

    Elemental composition of the solar atmosphere and in particular abundance bias of low and high First Ionization Potential (FIP) elements are a key tracer of the source regions of the solar wind. These abundances and their spatio-temporal variations, as well as the other plasma parameters , will be derived by the SPICE (Spectral Imaging of the Coronal Environment) EUV spectral imager on the upcoming Solar Orbiter mission. SPICE is designed to provide spectroheliograms (spectral images) using a core set of emission lines arising from ions of both low-FIP and high-FIP elements. These lines are formed over a wide range of temperatures, enabling the analysis of the different layers of the solar atmosphere. SPICE will use these spectroheliograms to produce dynamic composition maps of the solar atmosphere to be compared to in-situ measurements of the solar wind composition of the same elements (i.e. O, Ne, Mg, Fe). This will provide a tool to study the connectivity between the spacecraft (the Heliosphere) and the Sun. We will discuss the SPICE capabilities for such composition measurements.

  11. Near-infrared spectral imaging Michelson interferometer for astronomical applications

    NASA Technical Reports Server (NTRS)

    Wells, C. W.; Potter, A. E.; Morgan, T. H.

    1980-01-01

    The design and operation of an imaging Michelson interferometer-spectrometer used for near-infrared (0.8 micron to 2.5 microns) spectral imaging are reported. The system employs a rapid scan interferometer modified for stable low resolution (250/cm) performance and a 42 element PbS linear detector array. A microcomputer system is described which provides data acquisition, coadding, and Fourier transformation for near real-time presentation of the spectra of all 42 scene elements. The electronic and mechanical designs are discussed and telescope performance data presented.

  12. Spectrally optimal illuminations for diabetic retinopathy detection in retinal imaging

    NASA Astrophysics Data System (ADS)

    Bartczak, Piotr; Fält, Pauli; Penttinen, Niko; Ylitepsa, Pasi; Laaksonen, Lauri; Lensu, Lasse; Hauta-Kasari, Markku; Uusitalo, Hannu

    2017-01-01

    Retinal photography is a standard method for recording retinal diseases for subsequent analysis and diagnosis. However, the currently used white light or red-free retinal imaging does not necessarily provide the best possible visibility of different types of retinal lesions, important when developing diagnostic tools for handheld devices, such as smartphones. Using specifically designed illumination, the visibility and contrast of retinal lesions could be improved. In this study, spectrally optimal illuminations for diabetic retinopathy lesion visualization are implemented using a spectrally tunable light source based on digital micromirror device. The applicability of this method was tested in vivo by taking retinal monochrome images from the eyes of five diabetic volunteers and two non-diabetic control subjects. For comparison to existing methods, we evaluated the contrast of retinal images taken with our method and red-free illumination. The preliminary results show that the use of optimal illuminations improved the contrast of diabetic lesions in retinal images by 30-70%, compared to the traditional red-free illumination imaging.

  13. Single-shot ultrafast tomographic imaging by spectral multiplexing

    NASA Astrophysics Data System (ADS)

    Matlis, N. H.; Axley, A.; Leemans, W. P.

    2012-10-01

    Computed tomography has profoundly impacted science, medicine and technology by using projection measurements scanned over multiple angles to permit cross-sectional imaging of an object. The application of computed tomography to moving or dynamically varying objects, however, has been limited by the temporal resolution of the technique, which is set by the time required to complete the scan. For objects that vary on ultrafast timescales, traditional scanning methods are not an option. Here we present a non-scanning method capable of resolving structure on femtosecond timescales by using spectral multiplexing of a single laser beam to perform tomographic imaging over a continuous range of angles simultaneously. We use this technique to demonstrate the first single-shot ultrafast computed tomography reconstructions and obtain previously inaccessible structure and position information for laser-induced plasma filaments. This development enables real-time tomographic imaging for ultrafast science, and offers a potential solution to the challenging problem of imaging through scattering surfaces.

  14. Single-shot ultrafast tomographic imaging by spectral multiplexing.

    PubMed

    Matlis, N H; Axley, A; Leemans, W P

    2012-01-01

    Computed tomography has profoundly impacted science, medicine and technology by using projection measurements scanned over multiple angles to permit cross-sectional imaging of an object. The application of computed tomography to moving or dynamically varying objects, however, has been limited by the temporal resolution of the technique, which is set by the time required to complete the scan. For objects that vary on ultrafast timescales, traditional scanning methods are not an option. Here we present a non-scanning method capable of resolving structure on femtosecond timescales by using spectral multiplexing of a single laser beam to perform tomographic imaging over a continuous range of angles simultaneously. We use this technique to demonstrate the first single-shot ultrafast computed tomography reconstructions and obtain previously inaccessible structure and position information for laser-induced plasma filaments. This development enables real-time tomographic imaging for ultrafast science, and offers a potential solution to the challenging problem of imaging through scattering surfaces.

  15. Biomass estimator for NIR image with a few additional spectral band images taken from light UAS

    NASA Astrophysics Data System (ADS)

    Pölönen, Ilkka; Salo, Heikki; Saari, Heikki; Kaivosoja, Jere; Pesonen, Liisa; Honkavaara, Eija

    2012-05-01

    A novel way to produce biomass estimation will offer possibilities for precision farming. Fertilizer prediction maps can be made based on accurate biomass estimation generated by a novel biomass estimator. By using this knowledge, a variable rate amount of fertilizers can be applied during the growing season. The innovation consists of light UAS, a high spatial resolution camera, and VTT's novel spectral camera. A few properly selected spectral wavelengths with NIR images and point clouds extracted by automatic image matching have been used in the estimation. The spectral wavelengths were chosen from green, red, and NIR channels.

  16. Laser multi-spectral polarimetric diffuse-scatter imaging

    NASA Astrophysics Data System (ADS)

    Wang, Yang

    Laser multi-spectral polarimetric diffuse scatter (LAMPODS) imaging is an approach that maps an object intrinsic optical scattering properties rather than the scattered light intensity like in conventional imaging. The technique involves comprehensive measurements of the object scattering response function that is to be parameterized with respect to wavelength, polarization, and angular scattering distribution. The LAMPODS images are mappings of the derived parameters, which are more fundamental than conventional images. The LAMPODS imaging system was built based on a system architecture design configured similarly to an optical wireless network that allows multiple communication connections simultaneously among any number of transmitters and receivers. The imaging system was implemented into several sets of experimental apparatuses that can detect Stokes vectors of backward and forward scattered light with laser sources at seven near infrared (NIR) wavelengths and a continuous mid-infrared (mid-IR) spectral range for both macroscopic and microscopic scan imaging applications. The system components, such as transmitters, receivers, image scan unit, and the data acquisition module, were built and/or tested to match the system-design requirements, which involved many optical, opto-mechanical, electronic, and computer programming/interfacing techniques and skills. The experiments performed include the study on the LAMPODS capability with isolated aspects of scattering response, and the test of LAMPODS on uncontrolled subjects. With special-made targets, the results indicate that the LAMPODS system can distinguish consistently the four produced random surface roughnesses, regardless of the subjects? Spectroscopic signature, and can separate the spectroscopic features independently. Various natural and man-made targets were tested to challenge the LAMPODS system capability and found many interesting features regarding spectral response, polarimetric response, and

  17. Superharmonic imaging with chirp coded excitation: filtering spectrally overlapped harmonics.

    PubMed

    Harput, Sevan; McLaughlan, James; Cowell, David M J; Freear, Steven

    2014-11-01

    Superharmonic imaging improves the spatial resolution by using the higher order harmonics generated in tissue. The superharmonic component is formed by combining the third, fourth, and fifth harmonics, which have low energy content and therefore poor SNR. This study uses coded excitation to increase the excitation energy. The SNR improvement is achieved on the receiver side by performing pulse compression with harmonic matched filters. The use of coded signals also introduces new filtering capabilities that are not possible with pulsed excitation. This is especially important when using wideband signals. For narrowband signals, the spectral boundaries of the harmonics are clearly separated and thus easy to filter; however, the available imaging bandwidth is underused. Wideband excitation is preferable for harmonic imaging applications to preserve axial resolution, but it generates spectrally overlapping harmonics that are not possible to filter in time and frequency domains. After pulse compression, this overlap increases the range side lobes, which appear as imaging artifacts and reduce the Bmode image quality. In this study, the isolation of higher order harmonics was achieved in another domain by using the fan chirp transform (FChT). To show the effect of excitation bandwidth in superharmonic imaging, measurements were performed by using linear frequency modulated chirp excitation with varying bandwidths of 10% to 50%. Superharmonic imaging was performed on a wire phantom using a wideband chirp excitation. Results were presented with and without applying the FChT filtering technique by comparing the spatial resolution and side lobe levels. Wideband excitation signals achieved a better resolution as expected, however range side lobes as high as -23 dB were observed for the superharmonic component of chirp excitation with 50% fractional bandwidth. The proposed filtering technique achieved >50 dB range side lobe suppression and improved the image quality without

  18. Double-arm three-dimensional ion imaging apparatus for the study of ion pair channels in resonance enhanced multiphoton ionization

    NASA Astrophysics Data System (ADS)

    Poretskiy, M. S.; Chichinin, A. I.; Maul, C.; Gericke, K.-H.

    2016-02-01

    We present a novel experimental configuration for the full quantitative characterization of the multichannel resonance enhanced multiphoton ionization (REMPI) of small molecules in cases when the ion-pair dissociation channel is important. For this purpose, a double-arm time-of-flight mass spectrometer with three-dimensional (3D) ion imaging detectors at both arms is constructed. The REMPI of HCl molecules is used to examine the constructed setup. The apparatus allows us to perform simultaneous measurements of the 3D velocity vector distributions of positive (H+, HCl+, and Cl+) and negative (Cl-) photoions. The characterization consists of the determination of "two-photon absorption cross sections" for the process HCl(X)+2hν → HCl∗, one-photon absorption cross sections for subsequent processes HCl∗ + hν → HCl∗∗, and the probability of the subsequent non-adiabatic transition HCl∗∗ → HCl(B) → H+ + Cl-, which leads to ionic pairs. All these data should be obtained from the analysis of the dependencies of the number of ions on the laser energy. The full characterization of the laser beam and the knowledge of the ion detection probability are necessary parts of the analysis. Detailed knowledge of losses of produced ions in the mass spectrometer before detection requires understanding and characterization of such processes like electron emission from metallic grids under ion bombardment or charge transfer between positive ions and the metal surface of the grids, like Cl+ + (grid) → Cl-. These important phenomena from surface science are rarely discussed in the imaging literature, and here, we try to compensate for this shortcoming.

  19. Label-free multiphoton fluorescence imaging monitors metabolism in living primary human cells used for tissue engineering

    NASA Astrophysics Data System (ADS)

    Chen, Leng-Chun; Lloyd, William R.; Kuo, Shiuhyang; Marcelo, Cynthia L.; Feinberg, Stephen E.; Mycek, Mary-Ann

    2012-03-01

    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

  20. Thermal Infrared Spectral Imager for Airborne Science Applications

    NASA Technical Reports Server (NTRS)

    Johnson, William R.; Hook, Simon J.; Mouroulis, Pantazis; Wilson, Daniel W.; Gunapala, Sarath D.; Hill, Cory J.; Mumolo, Jason M.; Eng, Bjorn T.

    2009-01-01

    An airborne thermal hyperspectral imager is under development which utilizes the compact Dyson optical configuration and quantum well infrared photo detector (QWIP) focal plane array. The Dyson configuration uses a single monolithic prism-like grating design which allows for a high throughput instrument (F/1.6) with minimal ghosting, stray-light and large swath width. The configuration has the potential to be the optimal imaging spectroscopy solution for lighter-than-air (LTA) vehicles and unmanned aerial vehicles (UAV) due to its small form factor and relatively low power requirements. The planned instrument specifications are discussed as well as design trade-offs. Calibration testing results (noise equivalent temperature difference, spectral linearity and spectral bandwidth) and laboratory emissivity plots from samples are shown using an operational testbed unit which has similar specifications as the final airborne system. Field testing of the testbed unit was performed to acquire plots of apparent emissivity for various known standard minerals (such as quartz). A comparison is made using data from the ASTER spectral library.

  1. An Integrated Imaging Detector of Polarization and Spectral Content

    NASA Technical Reports Server (NTRS)

    Rust, D. M.; Thompson, K. E.

    1993-01-01

    A new type of image detector has been designed to simultaneously analyze the polarization of light at all picture elements in a scene. The Integrated Dual Imaging Detector (IDID) consists of a polarizing beamsplitter bonded to a charge-coupled device (CCD), with signal-analysis circuitry and analog-to-digital converters, all integrated on a silicon chip. It should be capable of 1:10(exp 4) polarization discrimination. The IDID should simplify the design and operation of imaging polarimeters and spectroscopic imagers used, for example, in atmospheric and solar research. Innovations in the IDID include (1) two interleaved 512 x 1024-pixel imaging arrays (one for each polarization plane); (2) large dynamic range (well depth of 10(exp 6) electrons per pixel); (3) simultaneous readout of both images at 10 million pixels per second each; (4) on-chip analog signal processing to produce polarization maps in real time; (5) on-chip 10-bit A/D conversion. When used with a lithium-niobate Fabry-Perot etalon or other color filter that can encode spectral information as polarization, the IDID can collect and analyze simultaneous images at two wavelengths. Precise photometric analysis of molecular or atomic concentrations in the atmosphere is one suggested application. When used in a solar telescope, the IDID will charge the polarization, which can then be converted to maps of the vector magnetic fields on the solar surface.

  2. Sparsity based denoising of spectral domain optical coherence tomography images

    PubMed Central

    Fang, Leyuan; Li, Shutao; Nie, Qing; Izatt, Joseph A.; Toth, Cynthia A.; Farsiu, Sina

    2012-01-01

    In this paper, we make contact with the field of compressive sensing and present a development and generalization of tools and results for reconstructing irregularly sampled tomographic data. In particular, we focus on denoising Spectral-Domain Optical Coherence Tomography (SDOCT) volumetric data. We take advantage of customized scanning patterns, in which, a selected number of B-scans are imaged at higher signal-to-noise ratio (SNR). We learn a sparse representation dictionary for each of these high-SNR images, and utilize such dictionaries to denoise the low-SNR B-scans. We name this method multiscale sparsity based tomographic denoising (MSBTD). We show the qualitative and quantitative superiority of the MSBTD algorithm compared to popular denoising algorithms on images from normal and age-related macular degeneration eyes of a multi-center clinical trial. We have made the corresponding data set and software freely available online. PMID:22567586

  3. Measuring Agarwood Formation Ratio Quantitatively by Fluorescence Spectral Imaging Technique.

    PubMed

    Huang, Botao; Nguyen, Duykien; Liu, Tianyi; Jiang, Kaibin; Tan, Jinfen; Liu, Chunxin; Zhao, Jing; Huang, Shaowei

    2015-01-01

    Agarwood is a kind of important and precious traditional Chinese medicine. With the decreasing of natural agarwood, artificial cultivation has become more and more important in recent years. Quantifying the formation of agarwood is an essential work which could provide information for guiding cultivation and controlling quality. But people only can judge the amount of agarwood qualitatively by experience before. Fluorescence multispectral imaging method is presented to measure the agarwood quantitatively in this paper. A spectral cube from 450 nm to 800 nm was captured under the 365 nm excitation sources. The nonagarwood, agarwood, and rotten wood in the same sample were distinguished based on analyzing the spectral cube. Then the area ratio of agarwood to the whole sample was worked out, which is the quantitative information of agarwood area percentage. To our knowledge, this is the first time that the formation of agarwood was quantified accurately and nondestructively.

  4. Measuring Agarwood Formation Ratio Quantitatively by Fluorescence Spectral Imaging Technique

    PubMed Central

    Huang, Botao; Nguyen, Duykien; Jiang, Kaibin; Tan, Jinfen; Liu, Chunxin; Zhao, Jing; Huang, Shaowei

    2015-01-01

    Agarwood is a kind of important and precious traditional Chinese medicine. With the decreasing of natural agarwood, artificial cultivation has become more and more important in recent years. Quantifying the formation of agarwood is an essential work which could provide information for guiding cultivation and controlling quality. But people only can judge the amount of agarwood qualitatively by experience before. Fluorescence multispectral imaging method is presented to measure the agarwood quantitatively in this paper. A spectral cube from 450 nm to 800 nm was captured under the 365 nm excitation sources. The nonagarwood, agarwood, and rotten wood in the same sample were distinguished based on analyzing the spectral cube. Then the area ratio of agarwood to the whole sample was worked out, which is the quantitative information of agarwood area percentage. To our knowledge, this is the first time that the formation of agarwood was quantified accurately and nondestructively. PMID:26089935

  5. Fluorescence spectral imaging of dihydroxyacetone on skin in vivo.

    PubMed

    Forest, Susan E; Grothaus, Jeff T; Ertel, Keith D; Rader, Charlie; Plante, Janyl

    2003-05-01

    Dihydroxyacetone (DHA) has been proposed as a potential alternative to dansyl chloride for use as a fluorescence marker on skin to assess stratum corneum turnover time in vivo. However, the fluorescence from DHA on skin has not been adequately studied. To address this void, a noninvasive, noncontact spectral imaging system is used to characterize the fluorescence spectrum of DHA on skin in vivo and to determine the optimal wavelengths over which to collect the DHA signal that minimizes the contributions from skin autofluorescence. The DHA-skin fluorescence signal dominates the 580-680 nm region of the visible spectrum when excited with ultraviolet radiation in the 320-400 nm wavelength region (UVA). An explanation of the time-dependent spectral features is proposed in terms of DHA polymerization and binding to skin.

  6. Image-based spectral transmission estimation using "sensitivity comparison".

    PubMed

    Nahavandi, Alireza Mahmoudi; Tehran, Mohammad Amani

    2017-01-20

    Although digital cameras have been used for spectral reflectance estimation, transmission measurement has rarely been considered in studies. This study presents a method named sensitivity comparison (SC) for spectral transmission estimation. The method needs neither a priori knowledge from the samples nor statistical information of a given reflectance dataset. As with spectrophotometers, the SC method needs one shot for calibration and another shot for measurement. The method exploits the sensitivity of the camera in the absence and presence of transparent colored objects for transmission estimation. 138 Kodak Wratten Gelatin filter transmissions were used for controlling the proposed method. Using modeling of the imaging system in different levels of noise, the performance of the proposed method was compared with a training-based Matrix R method. For checking the performance of the SC method in practice, 33 manmade colored transparent films were used in a conventional three-channel camera. The method generated promising results using different error metrics.

  7. Integrated coherent Raman scattering and multiphoton microscopy for label-free imaging of the dentin in the tooth

    NASA Astrophysics Data System (ADS)

    Wang, Zi; Zheng, Wei; Lin, Jian; Hsu, Chin-Ying; Huang, Zhiwei

    2014-02-01

    We report the implementation of a unique multimodal nonlinear optical microscopy (i.e., coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), third harmonic generation (THG) and two photon excitation fluorescence (TPEF)) platform for label-free imaging of dentin. A picosecond tunable laser together with an OPO is used as the excitation source for simultaneously multimodal imaging. CARS shows similar information as TPEF in dentin, but it has a higher sectioning performance than TPEF and thus it is a good alternative for TPEF. Microtubule structure is revealed nearby dentin enamel junction (DEJ) from the multimodal images. This work demonstrates that combining different nonlinear optical imaging modalities can provide new insights into the understanding of morphological structures and biochemical/biomolecular distributions of the dentine without the need of labeling.

  8. a High-Efficiency Fusion Method of Multi-Spectral Image and Panchromatic Image

    NASA Astrophysics Data System (ADS)

    Xue, X.; Wang, J. P.; Wang, H.; Xiang, F.

    2013-07-01

    With the development of modern remote sensing technology, a variety of earth observation satellites could continue to tremendously provide image data of different spatial resolution, time resolution, spectral resolution remote sensing, and the remote sensing data obtained is increasing with great capacity, which forms multi-source image pyramid in the same area. To play the advantages of a variety of remote sensing data, the application of remote sensing image fusion is a very important choice. When remote sensing data is large, fusion is large in computing capacity and time-consuming, so it is difficult to carry out rapid, real-time fusion. However, in some remote sensing applications, such as disaster prevention and relief quick, etc., timely fusion is required. Based on image fusion method of principal component analysis (PCA) and the advantage of parallel computing, a high-efficiency fusion method of multi-spectral image and panchromatic image is proposed. Beijing-1 Micro-satellite is a high-performance small satellite for earth observation,With Beijing-1 Micro-satellite remote sensing images as the experimental data, it is proved that good fusion results of multi-spectral image and panchromatic image can be obtained with the proposed method, and the fusion speed is also fast. At the same time, some measures of improving the efficiency of parallel image fusion are also discussed.

  9. SAR image segmentation using MSER and improved spectral clustering

    NASA Astrophysics Data System (ADS)

    Gui, Yang; Zhang, Xiaohu; Shang, Yang

    2012-12-01

    A novel approach is presented for synthetic aperture radar (SAR) image segmentation. By incorporating the advantages of maximally stable extremal regions (MSER) algorithm and spectral clustering (SC) method, the proposed approach provides effective and robust segmentation. First, the input image is transformed from a pixel-based to a region-based model by using the MSER algorithm. The input image after MSER procedure is composed of some disjoint regions. Then the regions are treated as nodes in the image plane, and a graph structure is applied to represent them. Finally, the improved SC is used to perform globally optimal clustering, by which the result of image segmentation can be generated. To avoid some incorrect partitioning when considering each region as one graph node, we assign different numbers of nodes to represent the regions according to area ratios among the regions. In addition, K-harmonic means instead of K-means is applied in the improved SC procedure in order to raise its stability and performance. Experimental results show that the proposed approach is effective on SAR image segmentation and has the advantage of calculating quickly.

  10. Segmentation of dynamic PET images with kinetic spectral clustering

    NASA Astrophysics Data System (ADS)

    Mouysset, S.; Zbib, H.; Stute, S.; Girault, J. M.; Charara, J.; Noailles, J.; Chalon, S.; Buvat, I.; Tauber, C.

    2013-10-01

    Segmentation is often required for the analysis of dynamic positron emission tomography (PET) images. However, noise and low spatial resolution make it a difficult task and several supervised and unsupervised methods have been proposed in the literature to perform the segmentation based on semi-automatic clustering of the time activity curves of voxels. In this paper we propose a new method based on spectral clustering that does not require any prior information on the shape of clusters in the space in which they are identified. In our approach, the p-dimensional data, where p is the number of time frames, is first mapped into a high dimensional space and then clustering is performed in a low-dimensional space of the Laplacian matrix. An estimation of the bounds for the scale parameter involved in the spectral clustering is derived. The method is assessed using dynamic brain PET images simulated with GATE and results on real images are presented. We demonstrate the usefulness of the method and its superior performance over three other clustering methods from the literature. The proposed approach appears as a promising pre-processing tool before parametric map calculation or ROI-based quantification tasks.

  11. Multiphoton Assisted Recombination

    NASA Astrophysics Data System (ADS)

    Shuman, E. S.; Jones, R. R.; Gallagher, T. F.

    2008-12-01

    We have observed multiphoton assisted recombination in the presence of a 38.8 GHz microwave field. Stimulated emission of up to ten microwave photons results in energy transfer from continuum electrons, enabling recombination. The maximum electron energy loss is far greater than the 2Up predicted by the standard “simpleman’s” model. The data are well reproduced by both an approximate analytic expression and numerical simulations in which the combined Coulomb and radiation fields are taken into account.

  12. Ultrahigh speed spectral/Fourier domain ophthalmic OCT imaging

    NASA Astrophysics Data System (ADS)

    Potsaid, Benjamin; Gorczynska, Iwona; Srinivasan, Vivek J.; Chen, Yueli; Liu, Jonathan; Jiang, James; Cable, Alex; Duker, Jay S.; Fujimoto, James G.

    2009-02-01

    Ultrahigh speed spectral / Fourier domain optical coherence tomography (OCT) imaging using a CMOS line scan camera with acquisition rates of 70,000 - 312,500 axial scans per second is investigated. Several design configurations are presented to illustrate trade-offs between acquisition speed, sensitivity, resolution and sensitivity roll-off performance. We demonstrate: extended imaging range and improved sensitivity roll-off at 70,000 axial scans per second , high speed and ultrahigh resolution imaging at 106,382 axial scans per second, and ultrahigh speed imaging at 250,000-312,500 axial scans per second. Each configuration is characterized through optical testing and the trade-offs demonstrated with in vivo imaging of the fovea and optic disk in the human retina. OCT fundus images constructed from 3D-OCT data acquired at 250,000 axial scans per second have no noticeable discontinuity of retinal features and show that there are minimal motion artifacts. The fine structures of the lamina cribrosa can be seen. Long cross sectional scans are acquired at 70,000 axial scans per second for imaging large areas of the retina, including the fovea and optic disk. Rapid repeated imaging of a small volume (4D-OCT) enables time resolved visualization of the capillary network surrounding the INL and may show individual red blood cells. The results of this study suggest that high speed CMOS cameras can achieve a significant improvement in performance for ophthalmic imaging. This promises to have a powerful impact in clinical applications by improving early diagnosis, reproducibility of measurements and enabling more sensitive assessment of disease progression or response to therapy.

  13. First multiphoton tomography of brain in man

    NASA Astrophysics Data System (ADS)

    König, Karsten; Kantelhardt, Sven R.; Kalasauskas, Darius; Kim, Ella; Giese, Alf

    2016-03-01

    We report on the first two-photon in vivo brain tissue imaging study in man. High resolution in vivo histology by multiphoton tomography (MPT) including two-photon FLIM was performed in the operation theatre during neurosurgery to evaluate the feasibility to detect label-free tumor borders with subcellular resolution. This feasibility study demonstrates, that MPT has the potential to identify tumor borders on a cellular level in nearly real-time.

  14. Superresolving multiphoton interferences with independent light sources.

    PubMed

    Oppel, S; Büttner, T; Kok, P; von Zanthier, J

    2012-12-07

    We propose to use multiphoton interferences from statistically independent light sources in combination with linear optical detection techniques to enhance the resolution in imaging. Experimental results with up to five independent thermal light sources confirm this approach to improve the spatial resolution. Since no involved quantum state preparation or detection is required, the experiment can be considered an extension of the Hanbury Brown-Twiss experiment for spatial intensity correlations of order N>2.

  15. Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal

    PubMed Central

    Ducourthial, Guillaume; Leclerc, Pierre; Mansuryan, Tigran; Fabert, Marc; Brevier, Julien; Habert, Rémi; Braud, Flavie; Batrin, Renaud; Vever-Bizet, Christine; Bourg-Heckly, Geneviève; Thiberville, Luc; Druilhe, Anne; Kudlinski, Alexandre; Louradour, Frédéric

    2015-01-01

    We present a two-photon microendoscope capable of in vivo label-free deep-tissue high-resolution fast imaging through a very long optical fiber. First, an advanced light-pulse spectro-temporal shaping device optimally precompensates for linear and nonlinear distortions occurring during propagation within the endoscopic fiber. This enables the delivery of sub-40-fs duration infrared excitation pulses at the output of 5 meters of fiber. Second, the endoscopic fiber is a custom-made double-clad polarization-maintaining photonic crystal fiber specifically designed to optimize the imaging resolution and the intrinsic luminescence backward collection. Third, a miniaturized fiber-scanner of 2.2 mm outer diameter allows simultaneous second harmonic generation (SHG) and two-photon excited autofluorescence (TPEF) imaging at 8 frames per second. This microendoscope’s transverse and axial resolutions amount respectively to 0.8 μm and 12 μm, with a field-of-view as large as 450 μm. This microendoscope’s unprecedented capabilities are validated during label-free imaging, ex vivo on various fixed human tissue samples, and in vivo on an anesthetized mouse kidney demonstrating an imaging penetration depth greater than 300 μm below the surface of the organ. The results reported in this manuscript confirm that nonlinear microendoscopy can become a valuable clinical tool for real-time in situ assessment of pathological states. PMID:26673905

  16. In vivo multiphoton nanosurgery on cortical neurons.

    PubMed

    Sacconi, Leonardo; O'Connor, Rodney P; Jasaitis, Audrius; Masi, Alessio; Buffelli, Mario; Pavone, Francesco S

    2007-01-01

    Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.

  17. Imaging of blood cells based on snapshot Hyper-Spectral Imaging systems

    NASA Astrophysics Data System (ADS)

    Robison, Christopher J.; Kolanko, Christopher; Bourlai, Thirimachos; Dawson, Jeremy M.

    2015-05-01

    Snapshot Hyper-Spectral imaging systems are capable of capturing several spectral bands simultaneously, offering coregistered images of a target. With appropriate optics, these systems are potentially able to image blood cells in vivo as they flow through a vessel, eliminating the need for a blood draw and sample staining. Our group has evaluated the capability of a commercial Snapshot Hyper-Spectral imaging system, the Arrow system from Rebellion Photonics, in differentiating between white and red blood cells on unstained blood smear slides. We evaluated the imaging capabilities of this hyperspectral camera; attached to a microscope at varying objective powers and illumination intensity. Hyperspectral data consisting of 25, 443x313 hyperspectral bands with ~3nm spacing were captured over the range of 419 to 494nm. Open-source hyper-spectral data cube analysis tools, used primarily in Geographic Information Systems (GIS) applications, indicate that white blood cells features are most prominent in the 428-442nm band for blood samples viewed under 20x and 50x magnification over a varying range of illumination intensities. These images could potentially be used in subsequent automated white blood cell segmentation and counting algorithms for performing in vivo white blood cell counting.

  18. Spectral-Spatial Classification of Hyperspectral Images Using Hierarchical Optimization

    NASA Technical Reports Server (NTRS)

    Tarabalka, Yuliya; Tilton, James C.

    2011-01-01

    A new spectral-spatial method for hyperspectral data classification is proposed. For a given hyperspectral image, probabilistic pixelwise classification is first applied. Then, hierarchical step-wise optimization algorithm is performed, by iteratively merging neighboring regions with the smallest Dissimilarity Criterion (DC) and recomputing class labels for new regions. The DC is computed by comparing region mean vectors, class labels and a number of pixels in the two regions under consideration. The algorithm is converged when all the pixels get involved in the region merging procedure. Experimental results are presented on two remote sensing hyperspectral images acquired by the AVIRIS and ROSIS sensors. The proposed approach improves classification accuracies and provides maps with more homogeneous regions, when compared to previously proposed classification techniques.

  19. Spectral Skyline Separation: Extended Landmark Databases and Panoramic Imaging

    PubMed Central

    Differt, Dario; Möller, Ralf

    2016-01-01

    Evidence from behavioral experiments suggests that insects use the skyline as a cue for visual navigation. However, changes of lighting conditions, over hours, days or possibly seasons, significantly affect the appearance of the sky and ground objects. One possible solution to this problem is to extract the “skyline” by an illumination-invariant classification of the environment into two classes, ground objects and sky. In a previous study (Insect models of illumination-invariant skyline extraction from UV (ultraviolet) and green channels), we examined the idea of using two different color channels available for many insects (UV and green) to perform this segmentation. We found out that for suburban scenes in temperate zones, where the skyline is dominated by trees and artificial objects like houses, a “local” UV segmentation with adaptive thresholds applied to individual images leads to the most reliable classification. Furthermore, a “global” segmentation with fixed thresholds (trained on an image dataset recorded over several days) using UV-only information is only slightly worse compared to using both the UV and green channel. In this study, we address three issues: First, to enhance the limited range of environments covered by the dataset collected in the previous study, we gathered additional data samples of skylines consisting of minerals (stones, sand, earth) as ground objects. We could show that also for mineral-rich environments, UV-only segmentation achieves a quality comparable to multi-spectral (UV and green) segmentation. Second, we collected a wide variety of ground objects to examine their spectral characteristics under different lighting conditions. On the one hand, we found that the special case of diffusely-illuminated minerals increases the difficulty to reliably separate ground objects from the sky. On the other hand, the spectral characteristics of this collection of ground objects covers well with the data collected in the skyline

  20. Spectral Skyline Separation: Extended Landmark Databases and Panoramic Imaging.

    PubMed

    Differt, Dario; Möller, Ralf

    2016-09-29

    Evidence from behavioral experiments suggests that insects use the skyline as a cue for visual navigation. However, changes of lighting conditions, over hours, days or possibly seasons, significantly affect the appearance of the sky and ground objects. One possible solution to this problem is to extract the "skyline" by an illumination-invariant classification of the environment into two classes, ground objects and sky. In a previous study (Insect models of illumination-invariant skyline extraction from UV (ultraviolet) and green channels), we examined the idea of using two different color channels available for many insects (UV and green) to perform this segmentation. We found out that for suburban scenes in temperate zones, where the skyline is dominated by trees and artificial objects like houses, a "local" UV segmentation with adaptive thresholds applied to individual images leads to the most reliable classification. Furthermore, a "global" segmentation with fixed thresholds (trained on an image dataset recorded over several days) using UV-only information is only slightly worse compared to using both the UV and green channel. In this study, we address three issues: First, to enhance the limited range of environments covered by the dataset collected in the previous study, we gathered additional data samples of skylines consisting of minerals (stones, sand, earth) as ground objects. We could show that also for mineral-rich environments, UV-only segmentation achieves a quality comparable to multi-spectral (UV and green) segmentation. Second, we collected a wide variety of ground objects to examine their spectral characteristics under different lighting conditions. On the one hand, we found that the special case of diffusely-illuminated minerals increases the difficulty to reliably separate ground objects from the sky. On the other hand, the spectral characteristics of this collection of ground objects covers well with the data collected in the skyline databases

  1. Feasibility study of a novel miniaturized spectral imaging system architecture in UAV surveillance

    NASA Astrophysics Data System (ADS)

    Liu, Shuyang; Zhou, Tao; Jia, Xiaodong; Cui, Hushan; Huang, Chengjun

    2016-01-01

    The spectral imaging technology is able to analysis the spectral and spatial geometric character of the target at the same time. To break through the limitation brought by the size, weight and cost of the traditional spectral imaging instrument, a miniaturized novel spectral imaging based on CMOS processing has been introduced in the market. This technology has enabled the possibility of applying spectral imaging in the UAV platform. In this paper, the relevant technology and the related possible applications have been presented to implement a quick, flexible and more detailed remote sensing system.

  2. Spectral photoplethysmographic imaging sensor fusion for enhanced heart rate detection

    NASA Astrophysics Data System (ADS)

    Amelard, Robert; Clausi, David A.; Wong, Alexander

    2016-03-01

    Continuous heart rate monitoring can provide important context for quantitative clinical assessment in scenarios such as long-term health monitoring and disability prevention. Photoplethysmographic imaging (PPGI) systems are particularly useful for such monitoring scenarios as contact-based devices pose problems related to comfort and mobility. Each pixel can be regarded as a virtual PPG sensor, thus enabling simultaneous measurements of multiple skin sites. Existing PPGI systems analyze temporal PPGI sensor uctuations related to hemodynamic pulsations across a region of interest to extract the blood pulse signal. However, due to spatially varying optical properties of the skin, the blood pulse signal may not be consistent across all PPGI sensors, leading to inaccurate heart rate monitoring. To increase the hemodynamic signal-to-noise ratio (SNR), we propose a novel spectral PPGI sensor fusion method for enhanced estimation of the true blood pulse signal. Motivated by the observation that PPGI sensors with high hemodynamic SNR exhibit a spectral energy peak at the heart rate frequency, an entropy-based fusion model was formulated to combine PPGI sensors based on the sensors' spectral energy distribution. The optical PPGI device comprised a near infrared (NIR) sensitive camera and an 850 nm LED. Spatially uniform irradiance was achieved by placing optical elements along the LED beam, providing consistent illumination across the skin area. Dual-mode temporally coded illumination was used to negate the temporal effect of ambient illumination. Experimental results show that the spectrally weighted PPGI method can accurately and consistently extract heart rate information where traditional region-based averaging fails.

  3. Studies of atmospheric molecules by multiphoton spectroscopy

    SciTech Connect

    Johnson, P.M.

    1991-10-01

    Carbon dioxide presents a great challenge to spectroscopy because of its propensity toward dissociation in all of its excited states. Multiphoton ionization spectroscopy is usually not applicable to the study of dissociating molecules because the dissociation competes effectively with ionization, resulting in no signal. We reasoned, however, that with high enough laser fluence, ionization could compete with dissociation in the longer lived states, exposing them for study from the continuous spectral background resulting from rapidly dissociating states. We describe the various spectroscopic and photophysical effects found through the multiphoton ionization and multiphoton photoelectron spectra. A recently developed variant of threshold ionization spectroscopy, usually called ZEKE, has shown a great deal of usefulness in providing the same information as traditional photoelectron spectroscopy but with higher resolution and much better signal-to-noise when using standard laboratory lasers. Threshold ionization techniques locate the states of an ion by scanning a light source across the ionization continuum of a neutral and somehow detecting when electrons are produced with no kinetic energy. We chose to develop our capabilities in threshold ionization spectroscopy using aromatic molecules because of their importance and because their electronic structure allows a pump-probe type of excitation scheme which avoids the use of vacuum ultraviolet laser beams. Among aromatics, the azines are noted for their small S{sub 1}-T{sub 1} energy gap which give them unique and interesting photophysical properties. We have continued our work on the multiphoton spectrum of metastable nitrogen produced by an electric discharge in supersonic beam. We have been able to assign more of the lines and simulated their rotational structure but many peaks remain unassigned.

  4. Gemstone spectral imaging for measuring adult bone mineral density

    PubMed Central

    Shao, Wei-Guang; Liu, Dian-Mei

    2016-01-01

    The present study aimed to detect the bone Ca2+ content of L3 vertebrae in adults by gemstone spectral computed tomography. In total, 235 patients were selected and divided into age groups of 10 years each. The scanning data were used to detect the water-based and Ca2+-based substance levels on the L3 vertebral cancellous bone images. The results indicated that there were significant differences in vertebral Ca2+-water and water-Ca2+ densities determined by gemstone spectral imaging (GSI) between males and females in subjects aged 50–59 years, 60–69 years, 70–79 years and ≥80 years (P<0.05). The ages of male and female participants were negatively correlated with vertebral Ca2+-water density (P<0.01) and water-Ca2+ density (P<0.01). In conclusion, GSI may be used as a novel method of measuring the vertebral adult bone mineral density. PMID:27703518

  5. Imaging spectral electrical properties of variably saturated porous media

    NASA Astrophysics Data System (ADS)

    Kelter, Matthias; Huisman, Johann A.; Kemna, Andreas; Zimmermann, Egon; Vereecken, Harry

    2013-04-01

    The spatial distribution of unsaturated hydraulic conductivity in the subsurface is of importance for hydrological modeling. Conventional methods to determine unsaturated hydraulic properties in the field are invasive and typically have a poor spatial resolution. In order to overcome these drawbacks, geophysical methods have received much attention in the last decades. Recent results of electrical impedance spectroscopy (EIS) on a range of saturated and unsaturated porous media revealed promising relationships between spectral electrical and hydraulic properties. Therefore, spectral electrical impedance tomography (EIT) is a promising method to image hydraulic properties in the subsurface. While this approach is emerging for aquifer characterization, unsaturated hydraulic properties have not yet been determined by EIT. In order to do so, a laboratory setup has been developed to perform controlled infiltration, drainage and stationary flow experiments on soil columns. A lysimeter with a height of 50 cm and a diameter of 22 cm is equipped with 40 electrodes and 4 tensiometers. An irrigation device at the top controlled by a peristaltic pump is used for a constant and homogeneous infiltration. Outflow is controlled by a suction plate at the bottom where an adjustable vacuum of up to 500 hPa can be applied. In a first measurement series, spectral EIT measurements were performed on a homogeneous sand column during stepwise drainage of the saturated porous medium using predefined pressure at the bottom. First results show that with decreasing water content the low frequency phase shift of complex electrical conductivity increases. This is consistent with previously reported EIS results. Calibrated relationships between electrical and hydraulic properties were used to convert the resulting electrical into hydraulic conductivity images.

  6. In vivo multiphoton tomography of skin cancer

    NASA Astrophysics Data System (ADS)

    König, Karsten; Riemann, Iris; Ehlers, Alexander; Buckle, Rainer; Dimitrow, Enrico; Kaatz, Martin; Fluhr, Joachim; Elsner, Peter

    2006-02-01

    The multiphoton tomograph DermaInspect was used to perform first clinical studies on the early non-invasive detection of skin cancer based on non-invasive optical sectioning of skin by two-photon autofluorescence and second harmonic generation. In particular, deep-tissue pigmented lesions -nevi- have been imaged with intracellular resolution using near infrared (NIR) femtosecond laser radiation. So far, more than 250 patients have been investigated. Cancerous tissues showed significant morphological differences compared to normal skin layers. In the case of malignant melanoma, the occurrence of luminescent melanocytes has been detected. Multiphoton tomography will become a novel non-invasive method to obtain high-resolution 3D optical biopsies for early cancer detection, treatment control, and in situ drug screening.

  7. Ratiometric spectral imaging for fast tumor detection and chemotherapy monitoring in vivo

    NASA Astrophysics Data System (ADS)

    Hwang, Jae Youn; Gross, Zeev; Gray, Harry B.; Medina-Kauwe, Lali K.; Farkas, Daniel L.

    2011-06-01

    We report a novel in vivo spectral imaging approach to cancer detection and chemotherapy assessment. We describe and characterize a ratiometric spectral imaging and analysis method and evaluate its performance for tumor detection and delineation by quantitatively monitoring the specific accumulation of targeted gallium corrole (HerGa) into HER2-positive (HER2 +) breast tumors. HerGa temporal accumulation in nude mice bearing HER2 + breast tumors was monitored comparatively by a. this new ratiometric imaging and analysis method; b. established (reflectance and fluorescence) spectral imaging; c. more commonly used fluorescence intensity imaging. We also tested the feasibility of HerGa imaging in vivo using the ratiometric spectral imaging method for tumor detection and delineation. Our results show that the new method not only provides better quantitative information than typical spectral imaging, but also better specificity than standard fluorescence intensity imaging, thus allowing enhanced in vivo outlining of tumors and dynamic, quantitative monitoring of targeted chemotherapy agent accumulation into them.

  8. Identifying pure urban image spectra using a learning urban image spectral archive (LUISA)

    NASA Astrophysics Data System (ADS)

    Jilge, Marianne; Heiden, Uta; Habermeyer, Martin; Mende, André; Juergens, Carsten

    2016-10-01

    In this study a learning urban image spectral archive (LUISA) has been developed, that overcomes the issue of an incomplete spectral library and can be used to derive scene-specific pure material spectra. It consists of a well described starting spectral library (LUISA-A) and a tool to derive scene-based pure surface material spectra (LUISA-T). The concept is based on a three-stage approach: (1) Comparing hyperspectral image spectra with LUISA-A spectra to identify scene-specific pure materials, (2) extracting unknown pure spectra based on spatial and spectral metrics and (3) provides the framework to implement new surface material spectra into LUISA-A. The spectral comparison is based on several similarity measures, followed by an object- and spectral-based ruleset to optimize and categorize potentially new pure spectra. The results show that the majority of pure surface materials could be identified using LUISA-A. Unknown spectra are composed of mixed pixels and real pure surface materials which could be distinguished by LUISA-T.

  9. Color camera computed tomography imaging spectrometer for improved spatial-spectral image accuracy

    NASA Technical Reports Server (NTRS)

    Wilson, Daniel W. (Inventor); Bearman, Gregory H. (Inventor); Johnson, William R. (Inventor)

    2011-01-01

    Computed tomography imaging spectrometers ("CTIS"s) having color focal plane array detectors are provided. The color FPA detector may comprise a digital color camera including a digital image sensor, such as a Foveon X3.RTM. digital image sensor or a Bayer color filter mosaic. In another embodiment, the CTIS includes a pattern imposed either directly on the object scene being imaged or at the field stop aperture. The use of a color FPA detector and the pattern improves the accuracy of the captured spatial and spectral information.

  10. Miniature Compressive Ultra-spectral Imaging System Utilizing a Single Liquid Crystal Phase Retarder

    PubMed Central

    August, Isaac; Oiknine, Yaniv; AbuLeil, Marwan; Abdulhalim, Ibrahim; Stern, Adrian

    2016-01-01

    Spectroscopic imaging has been proved to be an effective tool for many applications in a variety of fields, such as biology, medicine, agriculture, remote sensing and industrial process inspection. However, due to the demand for high spectral and spatial resolution it became extremely challenging to design and implement such systems in a miniaturized and cost effective manner. Using a Compressive Sensing (CS) setup based on a single variable Liquid Crystal (LC) retarder and a sensor array, we present an innovative Miniature Ultra-Spectral Imaging (MUSI) system. The LC retarder acts as a compact wide band spectral modulator. Within the framework of CS, a sequence of spectrally modulated images is used to recover ultra-spectral image cubes. Using the presented compressive MUSI system, we demonstrate the reconstruction of gigapixel spatio-spectral image cubes from spectral scanning shots numbering an order of magnitude less than would be required using conventional systems. PMID:27004447

  11. Spectral Properties and Dynamics of Gold Nanorods Revealed by EMCCD Based Spectral-Phasor Method

    PubMed Central

    Chen, Hongtao; Digman, Michelle A.

    2015-01-01

    Gold nanorods (NRs) with tunable plasmon-resonant absorption in the near-infrared region have considerable advantages over organic fluorophores as imaging agents. However, the luminescence spectral properties of NRs have not been fully explored at the single particle level in bulk due to lack of proper analytic tools. Here we present a global spectral phasor analysis method which allows investigations of NRs' spectra at single particle level with their statistic behavior and spatial information during imaging. The wide phasor distribution obtained by the spectral phasor analysis indicates spectra of NRs are different from particle to particle. NRs with different spectra can be identified graphically in corresponding spatial images with high spectral resolution. Furthermore, spectral behaviors of NRs under different imaging conditions, e.g. different excitation powers and wavelengths, were carefully examined by our laser-scanning multiphoton microscope with spectral imaging capability. Our results prove that the spectral phasor method is an easy and efficient tool in hyper-spectral imaging analysis to unravel subtle changes of the emission spectrum. Moreover, we applied this method to study the spectral dynamics of NRs during direct optical trapping and by optothermal trapping. Interestingly, spectral shifts were observed in both trapping phenomena. PMID:25684346

  12. Spectral properties and dynamics of gold nanorods revealed by EMCCD-based spectral phasor method.

    PubMed

    Chen, Hongtao; Gratton, Enrico; Digman, Michelle A

    2015-04-01

    Gold nanorods (NRs) with tunable plasmon-resonant absorption in the near-infrared region have considerable advantages over organic fluorophores as imaging agents due to their brightness and lack of photobleaching. However, the luminescence spectral properties of NRs have not been fully characterized at the single particle level due to lack of proper analytic tools. Here, we present a spectral phasor analysis method that allows investigations of NRs' spectra at single particle level showing the spectral variance and providing spatial information during imaging. The broad phasor distribution obtained by the spectral phasor analysis indicates that spectra of NRs are different from particle to particle. NRs with different spectra can be identified in images with high spectral resolution. The spectral behaviors of NRs under different imaging conditions, for example, different excitation powers and wavelengths, were revealed by our laser-scanning multiphoton microscope using a high-resolution spectrograph with imaging capability. Our results prove that the spectral phasor method is an easy and efficient tool in hyper-spectral imaging analysis to unravel subtle changes of the emission spectrum. We applied this method to study the spectral dynamics of NRs during direct optical trapping and by optothermal trapping. Interestingly, different spectral shifts were observed in both trapping phenomena.

  13. Spectral imager based on Fabry-Perot interferometer for Aalto-1 nanosatellite

    NASA Astrophysics Data System (ADS)

    Mannila, Rami; Näsilä, Antti; Viherkanto, Kai; Holmlund, Christer; Näkki, Ismo; Saari, Heikki

    2013-09-01

    The Aalto-1 is a 3U-cubesat project coordinated by Aalto University. The satellite, Aalto-1, will be mainly built by students as project assignments and thesis works. The Aalto-1 is planned to launch on 2014. VTT Technical Research Centre of Finland is developing the main Earth observation payload, a miniaturized spectral imager unit, for the satellite. The spectral imager unit contains a spectral imager, a visible RGB-camera and control electronics of the cameras. Detailed design of the spectral imager unit has been completed and assembly of the spectral imager unit will be done in the autumn 2013. The spectral imager is based on a tunable Fabry-Perot interferometer (FPI) accompanied by an RGB CMOS image sensor. The FPI consists of two highly reflective surfaces separated by a tunable air gap and it is based on a piezo-actuated structure. The piezo-actuated FPI uses three piezo-actuators and is controlled in a closed capacitive feedback loop. The spectral resolution of the imager will be 8-15 nm at full width at half maximum and it will operate in the wavelength range 500-900 nm. Imaging resolution of the spectral imager is 1024x1024 pixels and the focal length of the optics is 32 mm and F-number is 3.4. Mass of the spectral imager unit is approximately 600 grams, and dimensions are 97 mm x 97 mm x 48 mm.

  14. An excitation wavelength-scanning spectral imaging system for preclinical imaging

    NASA Astrophysics Data System (ADS)

    Leavesley, Silas; Jiang, Yanan; Patsekin, Valery; Rajwa, Bartek; Robinson, J. Paul

    2008-02-01

    Small-animal fluorescence imaging is a rapidly growing field, driven by applications in cancer detection and pharmaceutical therapies. However, the practical use of this imaging technology is limited by image-quality issues related to autofluorescence background from animal tissues, as well as attenuation of the fluorescence signal due to scatter and absorption. To combat these problems, spectral imaging and analysis techniques are being employed to separate the fluorescence signal from background autofluorescence. To date, these technologies have focused on detecting the fluorescence emission spectrum at a fixed excitation wavelength. We present an alternative to this technique, an imaging spectrometer that detects the fluorescence excitation spectrum at a fixed emission wavelength. The advantages of this approach include increased available information for discrimination of fluorescent dyes, decreased optical radiation dose to the animal, and ability to scan a continuous wavelength range instead of discrete wavelength sampling. This excitation-scanning imager utilizes an acousto-optic tunable filter (AOTF), with supporting optics, to scan the excitation spectrum. Advanced image acquisition and analysis software has also been developed for classification and unmixing of the spectral image sets. Filtering has been implemented in a single-pass configuration with a bandwidth (full width at half maximum) of 16nm at 550nm central diffracted wavelength. We have characterized AOTF filtering over a wide range of incident light angles, much wider than has been previously reported in the literature, and we show how changes in incident light angle can be used to attenuate AOTF side lobes and alter bandwidth. A new parameter, in-band to out-of-band ratio, was defined to assess the quality of the filtered excitation light. Additional parameters were measured to allow objective characterization of the AOTF and the imager as a whole. This is necessary for comparing the

  15. Multi-photon excitation microscopy

    PubMed Central

    Diaspro, Alberto; Bianchini, Paolo; Vicidomini, Giuseppe; Faretta, Mario; Ramoino, Paola; Usai, Cesare

    2006-01-01

    Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments. PMID:16756664

  16. Multi-photon excitation microscopy.

    PubMed

    Diaspro, Alberto; Bianchini, Paolo; Vicidomini, Giuseppe; Faretta, Mario; Ramoino, Paola; Usai, Cesare

    2006-06-06

    Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

  17. Multiphoton fluorescence microscopy in biology

    NASA Astrophysics Data System (ADS)

    Heikal, Ahmed A.; Webb, Watt W.

    2002-11-01

    The inherent advantages of nonlinear excitation make multiphoton fluorescence microscopy (MPFM) awell-suited imaging technique for extracting valuable information from turbid and thick biological samples. These advantages include high three-dimensional spatial resolution, large penetration depth, minimum out-of-focus cellular photodamage, and high signal-to-noise contrast. We have investigated the nonlinear spectroscopy of biologically important molecules such as NADH, flavins, and intrinsically fluorescent proteins. Fundamental understanding of the molecular spectroscopy and dynamics of these biomolecules is essential for advancing their applications in biological research. MPFM has been utilized for monitoring a large spectrum of biological processes including metabolic activity and exocytosis. We will discuss two-photon (2P) redox fluorescence microscopy of NADH, which gives a quantitative measure of the respiratory chain activity, thus allowing functional imaging of energy metabolism in neurons and native brain tissue. Finally, a rational design strategy, based on donor-acceptor-donor configuration, will be elucidated for fluorescent probes with large 2P-excitation cross-section. These dyes are water-soluble, yet possess a high affinity to organic phases with site-specific labeling and Ca+2 sensitivity (Kd ~ 350 nM). A brief account on the biological application of nanocrystals and second harmonic imaging will be reviewed.

  18. Spectrally Resolved Magnetic Resonance Imaging of the XenonBiosensor

    SciTech Connect

    Hilty, Christian; Lowery, Thomas; Wemmer, David; Pines, Alexander

    2005-07-15

    Due to its ability to non-invasively record images, as well as elucidate molecular structure, nuclear magnetic resonance is the method of choice for applications as widespread as chemical analysis and medical diagnostics. Its detection threshold is, however, limited by the small polarization of nuclear spins in even the highest available magnetic fields. This limitation can, under certain circumstances, be alleviated by using hyper-polarized substances. Xenon biosensors make use of the sensitivity gain of hyperpolarized xenon to provide magnetic resonance detection capability for a specific low-concentration target. They consist of a cryptophane cage, which binds one xenon atom, and which has been connected via a linker to a targeting moiety such as a ligand or antibody. Recent work has shown the possibility of using the xenon biosensor to detect small amounts of a substance in a heterogeneous environment by NMR. Here, we demonstrate that magnetic resonance (MR) provides the capability to obtain spectrally and spatially resolved images of the distribution of immobilized biosensor, opening the possibility for using the xenon biosensor for targeted imaging.

  19. Hyper-spectral imaging of aircraft exhaust plumes

    NASA Astrophysics Data System (ADS)

    Bowen, Spencer; Bradley, Kenneth; Gross, Kevin; Perram, Glen; Marciniak, Michael

    2008-10-01

    An imaging Fourier-transform spectrometer has been used to determine low spatial resolution temperature and chemical species concentration distributions of aircraft jet engine exhaust plumes. An overview of the imaging Fourier transform spectrometer and the methodology of the project is presented. Results to date are shared and future work is discussed. Exhaust plume data from a Turbine Technologies, LTD, SR-30 turbojet engine at three engine settings was collected using a Telops Field-portable Imaging Radiometric Spectrometer Technology Mid-Wave Extended (FIRST-MWE). Although the plume exhibited high temporal frequency fluctuations, temporal averaging of hyper-spectral data-cubes produced steady-state distributions, which, when co-added and Fourier transformed, produced workable spectra. These spectra were then reduced using a simplified gaseous effluent model to fit forward-modeled spectra obtained from the Line-By-Line Radiative Transfer Model (LBLRTM) and the high-resolution transmission (HITRAN) molecular absorption database to determine approximate temperature and concentration distributions. It is theorized that further development of the physical model will produce better agreement between measured and modeled data.

  20. Single-shot retinal imaging with AO spectral OCT

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Rha, Jungtae; Jonnal, Ravi S.; Miller, Donald T.

    2005-04-01

    We demonstrate for the first time an adaptive optics (AO) spectral OCT retina camera that acquires with unprecedented 3D resolution (2.9 μm lateral; 5.5 μm axial) single shot B-scans of the living human retina. The camera centers on a Michelson interferometer that consists of a superluminescent diode for line illuminating the subject's retinal; voice coil translator for controlling the optical path length of the reference channel; and an imaging spectrometer that is cascaded with a 12-bit area CCD array. The imaging spectrometer was designed with negligible off-axis aberrations and was constructed from stock optical components. AO was integrated into the detector channel of the interferometer and dynamically compensated for most of the ocular aberration across a 6 mm pupil. Short bursts of B-scans, with 100 Ascans each, were successfully acquired at 1 msec intervals. Camera sensitivity was found sufficient to detect reflections from all major retinal layers. Individual outer segments of photoreceptors at different retinal eccentricities were observed in vivo. Periodicity of the outer segments matched cone spacing as measured from AO flood illuminated images of the same patches of retina.

  1. High-resolution multimodal clinical multiphoton tomography of skin

    NASA Astrophysics Data System (ADS)

    König, Karsten

    2011-03-01

    This review focuses on multimodal multiphoton tomography based on near infrared femtosecond lasers. Clinical multiphoton tomographs for 3D high-resolution in vivo imaging have been placed into the market several years ago. The second generation of this Prism-Award winning High-Tech skin imaging tool (MPTflex) was introduced in 2010. The same year, the world's first clinical CARS studies have been performed with a hybrid multimodal multiphoton tomograph. In particular, non-fluorescent lipids and water as well as mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen has been imaged with submicron resolution in patients suffering from psoriasis. Further multimodal approaches include the combination of multiphoton tomographs with low-resolution wide-field systems such as ultrasound, optoacoustical, OCT, and dermoscopy systems. Multiphoton tomographs are currently employed in Australia, Japan, the US, and in several European countries for early diagnosis of skin cancer, optimization of treatment strategies, and cosmetic research including long-term testing of sunscreen nanoparticles as well as anti-aging products.

  2. Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing

    PubMed Central

    Sezgin, Erdinc; Waithe, Dominic; Bernardino de la Serna, Jorge; Eggeling, Christian

    2015-01-01

    Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane-embedded polarity-sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity-sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity-sensitive dyes C-Laurdan or Di-4-ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes. PMID:25755090

  3. Terahertz detectors for long wavelength multi-spectral imaging.

    SciTech Connect

    Lyo, Sungkwun Kenneth; Wanke, Michael Clement; Reno, John Louis; Shaner, Eric Arthur; Grine, Albert D.

    2007-10-01

    The purpose of this work was to develop a wavelength tunable detector for Terahertz spectroscopy and imaging. Our approach was to utilize plasmons in the channel of a specially designed field-effect transistor called the grating-gate detector. Grating-gate detectors exhibit narrow-linewidth, broad spectral tunability through application of a gate bias, and no angular dependence in their photoresponse. As such, if suitable sensitivity can be attained, they are viable candidates for Terahertz multi-spectral focal plane arrays. When this work began, grating-gate gate detectors, while having many promising characteristics, had a noise-equivalent power (NEP) of only 10{sup -5} W/{radical}Hz. Over the duration of this project, we have obtained a true NEP of 10{sup -8} W/{radical}Hz and a scaled NEP of 10{sup -9}W/{radical}Hz. The ultimate goal for these detectors is to reach a NEP in the 10{sup -9{yields}-10}W/{radical}Hz range; we have not yet seen a roadblock to continued improvement.

  4. Spectral Localization by Imaging Using Multi-Element Receiver Coils

    PubMed Central

    An, Li; Warach, Steven; Shen, Jun

    2010-01-01

    A new spectral localization technique for in vivo magnetic resonance spectroscopy (MRS) is introduced. Structural information extracted from anatomical imaging is utilized for defining compartments which provide the basis for spectral localization. Inherent spatial heterogeneity of multiple receiver coil elements is used along with optional phase encoding to resolve signals from different compartments. This technique allows a few compartmental spectra to be reconstructed from multi-channel data acquired with no or very few phase encoding steps, resulting in short scan time and high efficiency. Alternatively, this technique also allows a significant number of compartmental spectra to be reconstructed if sufficient phase encoding steps are used. A procedure is developed to semi-automatically generate a significant number of compartments of comparable sizes, which allows one to obtain spectra from small regions of interest with curvilinear shapes. This may be useful for obtaining spectra from relatively small stroke lesions or tumors. Phantom experiments and in vivo MRS of stroke patients have been performed to demonstrate this technique. PMID:21287595

  5. Research on method of geometry and spectral calibration of pushbroom dispersive hyperspectral imager

    NASA Astrophysics Data System (ADS)

    He, Zhiping; Shu, Rong; Wang, Jianyu

    2012-11-01

    Development and application of airborne and aerospace hyperspectral imager press for high precision geometry and spectral calibration of pixels of image cube. The research of geometry and spectral calibration of pushbroom hyperspectral imager, its target is giving the coordinate of angle field of view and center wavelength of each detect unit in focal plane detector of hyperspectral imager, and achieves the high precision, full field of view, full channel geometry and spectral calibration. It is importance for imaging quantitative and deep application of hyperspectal imager. The paper takes the geometry and spectral calibration of pushbroom dispersive hyperspectral imager as case study, and research on the constitution and analysis of imaging mathematical model. Aimed especially at grating-dispersive hyperspectral imaging, the specialty of the imaging mode and dispersive method has been concretely analyzed. Based on the analysis, the theory and feasible method of geometry and spectral calibration of dispersive hyperspectral imager is set up. The key technique has been solved is As follows: 1). the imaging mathematical model and feasible method of geometry and spectral calibration for full pixels of image cube has been set up, the feasibility of the calibration method has been analyzed. 2). the engineering model and method of the geometry and spectral calibration of pushbroom dispersive hyperspectral imager has been set up and the calibration equipment has been constructed, and the calibration precision has been analyzed.

  6. Unsupervised Learning of Cone Spectral Classes from Natural Images

    PubMed Central

    Benson, Noah C.; Manning, Jeremy R.; Brainard, David H.

    2014-01-01

    The first step in the evolution of primate trichromatic color vision was the expression of a third cone class not present in ancestral mammals. This observation motivates a fundamental question about the evolution of any sensory system: how is it possible to detect and exploit the presence of a novel sensory class? We explore this question in the context of primate color vision. We present an unsupervised learning algorithm capable of both detecting the number of spectral cone classes in a retinal mosaic and learning the class of each cone using the inter-cone correlations obtained in response to natural image input. The algorithm's ability to classify cones is in broad agreement with experimental evidence about functional color vision for a wide range of mosaic parameters, including those characterizing dichromacy, typical trichromacy, anomalous trichromacy, and possible tetrachromacy. PMID:24967877

  7. Unsupervised learning of cone spectral classes from natural images.

    PubMed

    Benson, Noah C; Manning, Jeremy R; Brainard, David H

    2014-06-01

    The first step in the evolution of primate trichromatic color vision was the expression of a third cone class not present in ancestral mammals. This observation motivates a fundamental question about the evolution of any sensory system: how is it possible to detect and exploit the presence of a novel sensory class? We explore this question in the context of primate color vision. We present an unsupervised learning algorithm capable of both detecting the number of spectral cone classes in a retinal mosaic and learning the class of each cone using the inter-cone correlations obtained in response to natural image input. The algorithm's ability to classify cones is in broad agreement with experimental evidence about functional color vision for a wide range of mosaic parameters, including those characterizing dichromacy, typical trichromacy, anomalous trichromacy, and possible tetrachromacy.

  8. Method for detection and imaging over a broad spectral range

    SciTech Connect

    Yefremenko, Volodymyr; Gordiyenko, Eduard; Pishko, legal representative, Olga; Novosad, Valentyn; Pishko, deceased; Vitalii

    2007-09-25

    A method of controlling the coordinate sensitivity in a superconducting microbolometer employs localized light, heating or magnetic field effects to form normal or mixed state regions on a superconducting film and to control the spatial location. Electron beam lithography and wet chemical etching were applied as pattern transfer processes in epitaxial Y--Ba--Cu--O films. Two different sensor designs were tested: (i) a 3 millimeter long and 40 micrometer wide stripe and (ii) a 1.25 millimeters long, and 50 micron wide meandering-like structure. Scanning the laser beam along the stripe leads to physical displacement of the sensitive area, and, therefore, may be used as a basis for imaging over a broad spectral range. Forming the superconducting film as a meandering structure provides the equivalent of a two-dimensional detector array. Advantages of this approach are simplicity of detector fabrication, and simplicity of the read-out process requiring only two electrical terminals.

  9. Iterative reconstruction of images from incomplete spectral data

    NASA Astrophysics Data System (ADS)

    Rhebergen, Jan B.; van den Berg, Peter M.; Habashy, Tarek M.

    1997-06-01

    In various branches of engineering and science, one is confronted with measurements resulting in incomplete spectral data. The problem of the reconstruction of an image from such a data set can be formulated in terms of an integral equation of the first kind. Consequently, this equation can be converted into an equivalent integral equation of the second kind which can be solved by a Neumann-type iterative method. It is shown that this Neumann expansion is an error-reducing method and that it is equivalent to the Papoulis - Gerchberg algorithm for band-limited signal extrapolation. The integral equation can also be solved by employing a conjugate gradient iterative scheme. Again, convergence of this scheme is demonstrated. Finally a number of illustrative numerical examples are presented and discussed.

  10. Fully Quantified Spectral Imaging Reveals in Vivo Membrane Protein Interactions

    PubMed Central

    King, Christopher; Stoneman, Michael; Raicu, Valerica; Hristova, Kalina

    2016-01-01

    Here we introduce the Fully Quantified Spectral Imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling. PMID:26787445

  11. Interferometric and nonlinear-optical spectral-imaging techniques for outer space and live cells

    NASA Astrophysics Data System (ADS)

    Itoh, Kazuyoshi

    2015-12-01

    Multidimensional signals such as the spectral images allow us to have deeper insights into the natures of objects. In this paper the spectral imaging techniques that are based on optical interferometry and nonlinear optics are presented. The interferometric imaging technique is based on the unified theory of Van Cittert-Zernike and Wiener-Khintchine theorems and allows us to retrieve a spectral image of an object in the far zone from the 3D spatial coherence function. The retrieval principle is explained using a very simple object. The promising applications to space interferometers for astronomy that are currently in progress will also be briefly touched on. An interesting extension of interferometric spectral imaging is a 3D and spectral imaging technique that records 4D information of objects where the 3D and spectral information is retrieved from the cross-spectral density function of optical field. The 3D imaging is realized via the numerical inverse propagation of the cross-spectral density. A few techniques suggested recently are introduced. The nonlinear optical technique that utilizes stimulated Raman scattering (SRS) for spectral imaging of biomedical targets is presented lastly. The strong signals of SRS permit us to get vibrational information of molecules in the live cell or tissue in real time. The vibrational information of unstained or unlabeled molecules is crucial especially for medical applications. The 3D information due to the optical nonlinearity is also the attractive feature of SRS spectral microscopy.

  12. First Radio Burst Imaging Observation From Mingantu Ultrawide Spectral Radioheliograph

    NASA Astrophysics Data System (ADS)

    Yan, Yihua; Chen, Linjie; Yu, Sijie; CSRH Team

    2015-08-01

    Radio imaging spectroscopy over wide range wavelength in dm/cm-bands will open new windows on solar flares and coronal mass ejections by tracing the radio emissions from accelerated electrons. The Chinese Spectral Radioheliograph (CSRH) with two arrays in 400MHz-2GHz /2-15GHz ranges with 64/532 frequency channels have been established in Mingantu Observing Station, Inner Mongolia of China, since 2013 and is in test observations now. CSRH is renamed as MUSER (Mingantu Ultrawide SpEctral Radioheliograph) after it's accomplishment We will introduce the progress and current status of CSRH. Some preliminary results of CSRH will be presented.On 11 Nov2014, the first burst event was registered by MUSER-I array at 400MHz-2GHz waveband. According to SGD event list there was a C-class flare peaked at 04:49UT in the disk center and the radio bursts around 04:22-04:24UT was attributed to this flare. However, MUSER-I image observation of the burst indicates that the radio burst peaked around 04:22UT was due to the eruption at the east limb of the Sun and connected to a CME appeared in that direction about 1 hour later. This demonstrate the importance of the spectroscopy observation of the solar radio burst.Acknowledgement: The CSRH team includes Wei Wang, Zhijun Chen, Fei Liu, Lihong Geng and Jian Zhang and CSRH project is supported by National Major Scientific Equipment R&D Project ZDYZ2009-3. The research was also supported by NSFC grants (11433006, 11221063), MOST grant (MOST2011CB811401), CAS Pilot-B Project (XDB09000000) and Marie Curie PIRSES- GA-295272-RADIOSUN.

  13. Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Sanchez, Washington Y.; Prow, Tarl W.; Sanchez, Washington H.; Grice, Jeffrey E.; Roberts, Michael S.

    2010-07-01

    Ex vivo human skin has been used extensively for cosmeceutical and drug delivery studies, transplantable skin allografts, or skin flaps. However, it has a half-life of a few days due to ischemic necrosis. Traditional methods of assessing viability can be time-consuming and provide limited metabolic information. Using multiphoton tomography and fluorescence lifetime imaging (MPT-FLIM) we assess ischemic necrosis of ex vivo skin by NAD(P)H autofluorescence intensity and fluorescence lifetime. Ex vivo skin is stored in the presence and absence of nutrient media (Dulbecco Modified Eagle Medium) at -20, 4, and 37 °C and room temperature over a 7-day time course to establish different rates of metabolic deterioration. At higher temperatures we observe a decrease in NAD(P)H autofluorescence, higher image noise, and a significant increase in the average fluorescence lifetime (τm) from ~1000 to 2000 ps. Additionally, significant distortions in NAD(P)H fluorescence lifetime histograms correspond to the reduction in autofluorescence. Skin kept at 4 °C, with or without media, showed the least change. Our findings suggest that MPT-FLIM enables useful noninvasive optical biopsies to monitor the metabolic state and deterioration of human skin for research and clinical purposes.

  14. Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Pena, A.-M.; Strupler, M.; Boulesteix, T.; Schanne-Klein, M.-C.

    2005-08-01

    We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin.

  15. Review of spectral imaging technology in biomedical engineering: achievements and challenges.

    PubMed

    Li, Qingli; He, Xiaofu; Wang, Yiting; Liu, Hongying; Xu, Dongrong; Guo, Fangmin

    2013-10-01

    Spectral imaging is a technology that integrates conventional imaging and spectroscopy to get both spatial and spectral information from an object. Although this technology was originally developed for remote sensing, it has been extended to the biomedical engineering field as a powerful analytical tool for biological and biomedical research. This review introduces the basics of spectral imaging, imaging methods, current equipment, and recent advances in biomedical applications. The performance and analytical capabilities of spectral imaging systems for biological and biomedical imaging are discussed. In particular, the current achievements and limitations of this technology in biomedical engineering are presented. The benefits and development trends of biomedical spectral imaging are highlighted to provide the reader with an insight into the current technological advances and its potential for biomedical research.

  16. Advances in renal (patho)physiology using multiphoton microscopy.

    PubMed

    Sipos, A; Toma, I; Kang, J J; Rosivall, L; Peti-Peterdi, J

    2007-11-01

    Multiphoton excitation fluorescence microscopy is a state-of-the-art confocal imaging technique ideal for deep optical sectioning of living tissues. It is capable of performing ultrasensitive, quantitative imaging of organ functions in health and disease with high spatial and temporal resolution which other imaging modalities cannot achieve. For more than a decade, multiphoton microscopy has been successfully used with various in vitro and in vivo experimental approaches to study many functions of different organs, including the kidney. This study focuses on recent advances in our knowledge of renal (patho)physiological processes made possible by the use of this imaging technology. Visualization of cellular variables like cytosolic calcium, pH, cell-to-cell communication and signal propagation, interstitial fluid flow in the juxtaglomerular apparatus (JGA), real-time imaging of tubuloglomerular feedback (TGF), and renin release mechanisms are reviewed. A brief summary is provided of kidney functions that can be measured by in vivo quantitative multiphoton imaging including glomerular filtration and permeability, concentration, dilution, and activity of the intrarenal renin-angiotensin system using this minimally invasive approach. New visual data challenge a number of existing paradigms in renal (patho)physiology. Also, quantitative imaging of kidney function with multiphoton microscopy has tremendous potential to eventually provide novel non-invasive diagnostic and therapeutic tools for future applications in clinical nephrology.

  17. Multimodal Spectral Imaging of Cells Using a Transmission Diffraction Grating on a Light Microscope

    PubMed Central

    Isailovic, Dragan; Xu, Yang; Copus, Tyler; Saraswat, Suraj; Nauli, Surya M.

    2011-01-01

    A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens. PMID:21639978

  18. Retinal Imaging of Infants on Spectral Domain Optical Coherence Tomography

    PubMed Central

    Vinekar, Anand; Mangalesh, Shwetha; Jayadev, Chaitra; Maldonado, Ramiro S.; Bauer, Noel; Toth, Cynthia A.

    2015-01-01

    Spectral domain coherence tomography (SD OCT) has become an important tool in the management of pediatric retinal diseases. It is a noncontact imaging device that provides detailed assessment of the microanatomy and pathology of the infant retina with a short acquisition time allowing office examination without the requirement of anesthesia. Our understanding of the development and maturation of the infant fovea has been enhanced by SD OCT allowing an in vivo assessment that correlates with histopathology. This has helped us understand the critical correlation of foveal development with visual potential in the first year of life and beyond. In this review, we summarize the recent literature on the clinical applications of SD OCT in studying the pathoanatomy of the infant macula, its ability to detect subclinical features, and its correlation with disease and vision. Retinopathy of prematurity and macular edema have been discussed in detail. The review also summarizes the current status of SD OCT in other infant retinal conditions, imaging the optic nerve, the choroid, and the retinal nerve fibre in infants and children, and suggests future areas of research. PMID:26221606

  19. Development and characterization of non-resonant multiphoton photoacoustic spectroscopy (NMPPAS) for brain tumor margining

    NASA Astrophysics Data System (ADS)

    Dahal, Sudhir

    ) and healthy tissue with over 99% accuracy. NMPPAS spectral features showed evident differences between tumor and healthy tissues, and ratiometric analysis ensured that only a few wavelengths could be used for excitation instead of using numerous wavelength excitations to create spectra. This process would significantly reduce the analysis time while maintaining the same degree of accuracy. Tissue phantoms were fabricated in order to characterize the properties of NMPPAS. Scattering particles were doped into the phantoms to simulate their light scattering properties to real tissues. This allowed for better control over shape, size, reproducibility and doping in the sample while maintaining the light-tissue interaction properties of real tissue. To make NMPPAS viable for clinical applications, the technique was characterized to determine the spatial (lateral and longitudinal) resolution, depth of penetration and its ability to image in three-dimension through layers of tissue. Both resolutions were determined to be near-cellular level resolution (50-70 microm), obtained initially with the aid of the technique of multiphoton fluorescence, and later verified using NMPPAS imaging. Additionally, the maximum depth of penetration and detection was determined to be about 1.4cm, making the technique extremely suitable to margin tumors from underlying tissues in the brain. The capability of NMPPAS to detect and image layers that lie beneath other structures and blood vessels was also investigated. Three-dimensional images were obtained for the first time using NMPPAS. The images were obtained from different depths and structures were imaged through other layers of existing structures in the sample. This verified that NMPPAS was capable of detecting and imaging structures that lie embedded within the tissues. NMPPAS images of embedded structures were also obtained with the presence of hemoglobin, which is potentially the largest source of background in blood-perfused tissues

  20. Semi-supervised segmentation of multispectral remote sensing image based on spectral clustering

    NASA Astrophysics Data System (ADS)

    Zhang, Xiangrong; Wang, Ting; Jiao, Licheng; Yang, Chun

    2009-10-01

    In this paper, a new multi-spectral remote sensing image segmentation method based on multi-parameter semi-supervised spectral clustering (STS3C) is proposed. Two types of instance-level constraints: must-link and cannot-link are incorporated into spectral cluster to construct semi-supervised spectral clustering in which the self-tuning parameter is applied to avoid the selection of the scaling parameter. Further, when STS3C is applied to multi-spectral remote sensing image segmentation, the uniform sampling technique combined with nearest neighbor rule is used to reduce the computation complexity. Segmentation results show that STS3C outperforms the semi-supervised spectral clustering with fixed parameter and the well-known clustering methods including k-means and FCM in multi-spectral remote sensing image segmentation.

  1. Temporal and spectral imaging with micro-CT

    SciTech Connect

    Johnston, Samuel M.; Johnson, G. Allan; Badea, Cristian T.

    2012-08-15

    can be used to segment regions containing iodinated blood and compute measures of cardiac function. Conclusions: We believe this combined spectral and temporal imaging technique will be useful for future studies of cardiopulmonary disease in small animals.

  2. Software defined multi-spectral imaging for Arctic sensor networks

    NASA Astrophysics Data System (ADS)

    Siewert, Sam; Angoth, Vivek; Krishnamurthy, Ramnarayan; Mani, Karthikeyan; Mock, Kenrick; Singh, Surjith B.; Srivistava, Saurav; Wagner, Chris; Claus, Ryan; Vis, Matthew Demi

    2016-05-01

    Availability of off-the-shelf infrared sensors combined with high definition visible cameras has made possible the construction of a Software Defined Multi-Spectral Imager (SDMSI) combining long-wave, near-infrared and visible imaging. The SDMSI requires a real-time embedded processor to fuse images and to create real-time depth maps for opportunistic uplink in sensor networks. Researchers at Embry Riddle Aeronautical University working with University of Alaska Anchorage at the Arctic Domain Awareness Center and the University of Colorado Boulder have built several versions of a low-cost drop-in-place SDMSI to test alternatives for power efficient image fusion. The SDMSI is intended for use in field applications including marine security, search and rescue operations and environmental surveys in the Arctic region. Based on Arctic marine sensor network mission goals, the team has designed the SDMSI to include features to rank images based on saliency and to provide on camera fusion and depth mapping. A major challenge has been the design of the camera computing system to operate within a 10 to 20 Watt power budget. This paper presents a power analysis of three options: 1) multi-core, 2) field programmable gate array with multi-core, and 3) graphics processing units with multi-core. For each test, power consumed for common fusion workloads has been measured at a range of frame rates and resolutions. Detailed analyses from our power efficiency comparison for workloads specific to stereo depth mapping and sensor fusion are summarized. Preliminary mission feasibility results from testing with off-the-shelf long-wave infrared and visible cameras in Alaska and Arizona are also summarized to demonstrate the value of the SDMSI for applications such as ice tracking, ocean color, soil moisture, animal and marine vessel detection and tracking. The goal is to select the most power efficient solution for the SDMSI for use on UAVs (Unoccupied Aerial Vehicles) and other drop

  3. Fast Multispectral Imaging by Spatial Pixel-Binning and Spectral Unmixing.

    PubMed

    Pan, Zhi-Wei; Shen, Hui-Liang; Li, Chunguang; Chen, Shu-Jie; Xin, John H

    2016-08-01

    Multispectral imaging system is of wide application in relevant fields for its capability in acquiring spectral information of scenes. Its limitation is that, due to the large number of spectral channels, the imaging process can be quite time-consuming when capturing high-resolution (HR) multispectral images. To resolve this limitation, this paper proposes a fast multispectral imaging framework based on the image sensor pixel-binning and spectral unmixing techniques. The framework comprises a fast imaging stage and a computational reconstruction stage. In the imaging stage, only a few spectral images are acquired in HR, while most spectral images are acquired in low resolution (LR). The LR images are captured by applying pixel binning on the image sensor, such that the exposure time can be greatly reduced. In the reconstruction stage, an optimal number of basis spectra are computed and the signal-dependent noise statistics are estimated. Then the unknown HR images are efficiently reconstructed by solving a closed-form cost function that models the spatial and spectral degradations. The effectiveness of the proposed framework is evaluated using real-scene multispectral images. Experimental results validate that, in general, the method outperforms the state of the arts in terms of reconstruction accuracy, with additional 20× or more improvement in computational efficiency.

  4. Multiphoton ionization of Uracil

    NASA Astrophysics Data System (ADS)

    Prieto, Eladio; Martinez, Denhi; Guerrero, Alfonso; Alvarez, Ignacio; Cisneros, Carmen

    2016-05-01

    Multiphoton ionization and dissociation of Uracil using a Reflectron time of flight spectrometer was performed along with radiation from the second harmonic of a Nd:YAG laser. Uracil is one of the four nitrogen bases that belong to RNA. The last years special interest has been concentrated on the study of the effects under UV radiation in nucleic acids1 and also in the role that this molecule could have played in the origin and development of life on our planet.2 The MPI mass spectra show that the presence and intensity of the resulting ions strongly depend on the density power. The identification of the ions in the mass spectra is presented. The results are compared with those obtained in other laboratories under different experimental conditions and some of them show partial agreement.3 The present work was supported by CONACYT-Mexico Grant 165410 and DGAPA UNAM Grant IN101215 and IN102613.

  5. The Spectral Image Processing System (SIPS) - Interactive visualization and analysis of imaging spectrometer data

    NASA Technical Reports Server (NTRS)

    Kruse, F. A.; Lefkoff, A. B.; Boardman, J. W.; Heidebrecht, K. B.; Shapiro, A. T.; Barloon, P. J.; Goetz, A. F. H.

    1993-01-01

    The Center for the Study of Earth from Space (CSES) at the University of Colorado, Boulder, has developed a prototype interactive software system called the Spectral Image Processing System (SIPS) using IDL (the Interactive Data Language) on UNIX-based workstations. SIPS is designed to take advantage of the combination of high spectral resolution and spatial data presentation unique to imaging spectrometers. It streamlines analysis of these data by allowing scientists to rapidly interact with entire datasets. SIPS provides visualization tools for rapid exploratory analysis and numerical tools for quantitative modeling. The user interface is X-Windows-based, user friendly, and provides 'point and click' operation. SIPS is being used for multidisciplinary research concentrating on use of physically based analysis methods to enhance scientific results from imaging spectrometer data. The objective of this continuing effort is to develop operational techniques for quantitative analysis of imaging spectrometer data and to make them available to the scientific community prior to the launch of imaging spectrometer satellite systems such as the Earth Observing System (EOS) High Resolution Imaging Spectrometer (HIRIS).

  6. Application of multivariate statistical analysis to STEM X-ray spectral images: interfacial analysis in microelectronics.

    PubMed

    Kotula, Paul G; Keenan, Michael R

    2006-12-01

    Multivariate statistical analysis methods have been applied to scanning transmission electron microscopy (STEM) energy-dispersive X-ray spectral images. The particular application of the multivariate curve resolution (MCR) technique provides a high spectral contrast view of the raw spectral image. The power of this approach is demonstrated with a microelectronics failure analysis. Specifically, an unexpected component describing a chemical contaminant was found, as well as a component consistent with a foil thickness change associated with the focused ion beam specimen preparation process. The MCR solution is compared with a conventional analysis of the same spectral image data set.

  7. Methodology for diagnosing of skin cancer on images of dermatologic spots by spectral analysis.

    PubMed

    Guerra-Rosas, Esperanza; Álvarez-Borrego, Josué

    2015-10-01

    In this paper a new methodology for the diagnosing of skin cancer on images of dermatologic spots using image processing is presented. Currently skin cancer is one of the most frequent diseases in humans. This methodology is based on Fourier spectral analysis by using filters such as the classic, inverse and k-law nonlinear. The sample images were obtained by a medical specialist and a new spectral technique is developed to obtain a quantitative measurement of the complex pattern found in cancerous skin spots. Finally a spectral index is calculated to obtain a range of spectral indices defined for skin cancer. Our results show a confidence level of 95.4%.

  8. Multimodal near infrared spectral imaging as an exploratory tool for dysplastic esophageal lesion identification

    NASA Astrophysics Data System (ADS)

    Lieber, Chad A.; Urayama, Shiro; Rahim, Nazir; Tu, Raymond; Saroufeem, Ramez; Reubner, Boris; Demos, Stavros G.

    2006-03-01

    We explore nine different combinations of fluorescence, light scattering, and polarization spectral imaging approaches in the near-infrare spectral region toward the diagnosis of pathologic and normal esophageal lesions. The combinations of all the imaging techniques were evaluated for maximal sensitivity and specificity. The results suggest that this multimodal approach is capable of highly accurate detection of the presence of pathologic tissue.

  9. Multifocal multiphoton microscopy with adaptive optical correction

    NASA Astrophysics Data System (ADS)

    Coelho, Simao; Poland, Simon; Krstajic, Nikola; Li, David; Monypenny, James; Walker, Richard; Tyndall, David; Ng, Tony; Henderson, Robert; Ameer-Beg, Simon

    2013-02-01

    Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled MegaFLI. Beam parallelization performed via a 3D Gerchberg-Saxton (GS) algorithm using a Spatial Light Modulator (SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction. Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.

  10. Spectral analysis for evaluation of myocardial tracers for medical imaging

    SciTech Connect

    Huesman, Ronald H.; Reutter, Bryan W.; Marshall, Robert C.

    2000-10-11

    Kinetic analysis of dynamic tracer data is performed with the goal of evaluating myocardial radiotracers for cardiac nuclear medicine imaging. Data from experiments utilizing the isolated rabbit heart model are acquired by sampling the venous blood after introduction of a tracer of interest and a reference tracer. We have taken the approach that the kinetics are properly characterized by an impulse response function which describes the difference between the reference molecule (which does not leave the vasculature) and the molecule of interest which is transported across the capillary boundary and is made available to the cell. Using this formalism we can model the appearance of the tracer of interest in the venous output of the heart as a convolution of the appearance of the reference tracer with the impulse response. In this work we parameterize the impulse response function as the sum of a large number of exponential functions whose predetermined decay constants form a spectrum, and each is required only to have a nonnegative coefficient. This approach, called spectral analysis, has the advantage that it allows conventional compartmental analysis without prior knowledge of the number of compartments which the physiology may require or which the data will support.

  11. Modeling spectrally varying resolution in broadband imaging systems

    NASA Astrophysics Data System (ADS)

    Cota, Stephen A.; Schnee, Scott L.; Loverro, Adam L.

    2015-09-01

    The remote sensing system engineering process often makes use of modeling and simulation tools to flow down specifications to subsystems and components, and/or to predict performance given a particular set of defined capabilities. A persistent question in the development and use of such tools is that of appropriate level of fidelity. In this paper we look at one problem area encountered in the simulation of panchromatic and other broadband imaging systems, that of accounting for spectrally varying resolution over the band. An established method for treating this variation is that of the polychromatic optical transfer function (OTF), but this technique imposes a measure of complexity on the simulation tool software architecture, as well as on users who must subsequently interact with it. We present a methodology for assessing the required level of fidelity for this problem and show that under some conditions it appears possible to forgo the polychromatic OTF formalism, or else to treat it with less than full rigor, with minimal loss in accuracy.

  12. Quantitative method to assess caries via fluorescence imaging from the perspective of autofluorescence spectral analysis

    NASA Astrophysics Data System (ADS)

    Chen, Q. G.; Zhu, H. H.; Xu, Y.; Lin, B.; Chen, H.

    2015-08-01

    A quantitative method to discriminate caries lesions for a fluorescence imaging system is proposed in this paper. The autofluorescence spectral investigation of 39 teeth samples classified by the International Caries Detection and Assessment System levels was performed at 405 nm excitation. The major differences in the different caries lesions focused on the relative spectral intensity range of 565-750 nm. The spectral parameter, defined as the ratio of wavebands at 565-750 nm to the whole spectral range, was calculated. The image component ratio R/(G + B) of color components was statistically computed by considering the spectral parameters (e.g. autofluorescence, optical filter, and spectral sensitivity) in our fluorescence color imaging system. Results showed that the spectral parameter and image component ratio presented a linear relation. Therefore, the image component ratio was graded as <0.66, 0.66-1.06, 1.06-1.62, and >1.62 to quantitatively classify sound, early decay, established decay, and severe decay tissues, respectively. Finally, the fluorescence images of caries were experimentally obtained, and the corresponding image component ratio distribution was compared with the classification result. A method to determine the numerical grades of caries using a fluorescence imaging system was proposed. This method can be applied to similar imaging systems.

  13. Short-wave infrared (SWIR) spectral imager based on Fabry-Perot interferometer for remote sensing

    NASA Astrophysics Data System (ADS)

    Mannila, Rami; Holmlund, Christer; Ojanen, Harri J.; Näsilä, Antti; Saari, Heikki

    2014-10-01

    VTT Technical Research Centre of Finland has developed a spectral imager for short-wave infrared (SWIR) wavelength range. The spectral imager is based on a tunable Fabry-Perot interferometer (FPI) accompanied by a commercial InGaAs Camera. The FPI consists of two dielectric coated mirrors separated by a tunable air gap. Tuning of the air gap tunes also transmitted wavelength and therefore FPI acts as a tunable band bass filter. The FPI is piezo-actuated and it uses three piezo-actuators in a closed capacitive feedback loop for air gap tuning. The FPI has multiple order transmission bands, which limit free spectral range. Therefore spectral imager contains two FPI in a stack, to make possible to cover spectral range of 1000 - 1700 nm. However, in the first tests imager was used with one FPI and spectral range was limited to 1100-1600 nm. The spectral resolution of the imager is approximately 15 nm (FWHM). Field of view (FOV) across the flight direction is 30 deg. Imaging resolution of the spectral imager is 256 x 320 pixels. The focal length of the optics is 12 mm and F-number is 3.2. This imager was tested in summer 2014 in an unmanned aerial vehicle (UAV) and therefore a size and a mass of the imager were critical. Total mass of the imager is approximately 1200 grams. In test campaign the spectral imager will be used for forest and agricultural imaging. In future, because results of the UAV test flights are promising, this technology can be applied to satellite applications also.

  14. [The hyperspectral camera side-scan geometric imaging in any direction considering the spectral mixing].

    PubMed

    Wang, Shu-Min; Zhang, Ai-Wu; Hu, Shao-Xing; Sun, Wei-Dong

    2014-07-01

    In order to correct the image distortion in the hyperspectral camera side-scan geometric Imaging, the image pixel geo-referenced algorithm was deduced in detail in the present paper, which is suitable to the linear push-broom camera side-scan imaging on the ground in any direction. It takes the orientation of objects in the navigation coordinates system into account. Combined with the ground sampling distance of geo-referenced image and the area of push broom imaging, the general process of geo-referenced image divided into grids is also presented. The new image rows and columns will be got through the geo-referenced image area dividing the ground sampling distance. Considering the error produced by round rule in the pixel grids generated progress, and the spectral mixing problem caused by traditional direct spectral sampling method in the process of image correction, the improved spectral sampling method based on the weighted fusion method was proposed. It takes the area proportion of adjacent pixels in the new generated pixel as coefficient and then the coefficients are normalized to avoid the spectral overflow. So the new generated pixel is combined with the geo-referenced adjacent pixels spectral. Finally the amounts of push-broom imaging experiments were taken on the ground, and the distortion images were corrected according to the algorithm proposed above. The results show that the linear image distortion correction algorithm is valid and robust. At the same time, multiple samples were selected in the corrected images to verify the spectral data. The results indicate that the improved spectral sampling method is better than the direct spectral sampling algorithm. It provides reference for the application of similar productions on the ground.

  15. Electron Vortices in Femtosecond Multiphoton Ionization

    NASA Astrophysics Data System (ADS)

    Pengel, D.; Kerbstadt, S.; Johannmeyer, D.; Englert, L.; Bayer, T.; Wollenhaupt, M.

    2017-02-01

    Multiphoton ionization of potassium atoms with a sequence of two counter-rotating circularly polarized femtosecond laser pulses produces vortex-shaped photoelectron momentum distributions in the polarization plane describing Archimedean spirals. The pulse sequences are produced by polarization shaping and the three-dimensional photoelectron distributions are tomographically reconstructed from velocity map imaging measurements. We show that perturbative ionization leads to electron vortices with c6 rotational symmetry. A change from c6 to c4 rotational symmetry of the vortices is demonstrated for nonperturbative interaction.

  16. Spectral images browsing using principal component analysis and set partitioning in hierarchical tree

    NASA Astrophysics Data System (ADS)

    Ma, Long; Zhao, Deping

    2011-12-01

    Spectral imaging technology have been used mostly in remote sensing, but have recently been extended to new area requiring high fidelity color reproductions like telemedicine, e-commerce, etc. These spectral imaging systems are important because they offer improved color reproduction quality not only for a standard observer under a particular illuminantion, but for any other individual exhibiting normal color vision capability under another illuminantion. A possibility for browsing of the archives is needed. In this paper, the authors present a new spectral image browsing architecture. The architecture for browsing is expressed as follow: (1) The spectral domain of the spectral image is reduced with the PCA transform. As a result of the PCA transform the eigenvectors and the eigenimages are obtained. (2) We quantize the eigenimages with the original bit depth of spectral image (e.g. if spectral image is originally 8bit, then quantize eigenimage to 8bit), and use 32bit floating numbers for the eigenvectors. (3) The first eigenimage is lossless compressed by JPEG-LS, the other eigenimages were lossy compressed by wavelet based SPIHT algorithm. For experimental evalution, the following measures were used. We used PSNR as the measurement for spectral accuracy. And for the evaluation of color reproducibility, ΔE was used.here standard D65 was used as a light source. To test the proposed method, we used FOREST and CORAL spectral image databases contrain 12 and 10 spectral images, respectively. The images were acquired in the range of 403-696nm. The size of the images were 128*128, the number of bands was 40 and the resolution was 8 bits per sample. Our experiments show the proposed compression method is suitable for browsing, i.e., for visual purpose.

  17. Compressive spectral polarization imaging by a pixelized polarizer and colored patterned detector.

    PubMed

    Fu, Chen; Arguello, Henry; Sadler, Brian M; Arce, Gonzalo R

    2015-11-01

    A compressive spectral and polarization imager based on a pixelized polarizer and colored patterned detector is presented. The proposed imager captures several dispersed compressive projections with spectral and polarization coding. Stokes parameter images at several wavelengths are reconstructed directly from 2D projections. Employing a pixelized polarizer and colored patterned detector enables compressive sensing over spatial, spectral, and polarization domains, reducing the total number of measurements. Compressive sensing codes are specially designed to enhance the peak signal-to-noise ratio in the reconstructed images. Experiments validate the architecture and reconstruction algorithms.

  18. Importance of the texture features in a query from a spectral image database

    NASA Astrophysics Data System (ADS)

    Kohonen, Oili; Hauta-Kasari, Markku

    2006-01-01

    A new, semantically meaningful technique for querying the images from a spectral image database is proposed. The technique is based on the use of both color- and texture features. The color features are calculated from spectral images by using the Self-Organizing Map (SOM) when methods of Gray Level Co-occurrence Matrix (GLCM) and Local Binary Pattern (LBP) are used for constructing the texture features. The importance of texture features in a querying is seen in experimental results, which are given by using a real spectral image database. Also the differences between the results gained by the use of co-occurrence matrix and LBP are introduced.

  19. The characteristic analysis of spectral image for cabbage leaves damaged by diamondback moth pests

    NASA Astrophysics Data System (ADS)

    Lin, Li-bo; Li, Hong-ning; Cao, Peng-fei; Qin, Feng; Yang, Shu-ming; Feng, Jie

    2015-02-01

    Cabbage growth and health diagnosis are important parts for cabbage fine planting, spectral imaging technology with the advantages of obtaining spectrum and space information of the target at the same time, which has become a research hotspot at home and abroad. The experiment measures the reflection spectrum at different stages using liquid crystal tunable filter (LCTF) and monochromatic CMOS camera composed of spectral imaging system for cabbage leaves damaged by diamondback moth pests, and analyzes its feature bands and the change of spectral parameters. The study shows that the feature bands of cabbage leaves damaged by diamondback moth pests have a tendency to blue light direction, the red edge towards blue shift, and red valley raising in spectral characteristic parameters, which have a good indication in diagnosing the extent of cabbage damaged by pests. Therefore, it has a unique advantage of monitoring the cabbage leaves damaged by diamondback moth pests by combinating feature bands and spectral characteristic parameters in spectral imaging technology.

  20. Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

    PubMed

    Luo, Yuan; Gelsinger-Austin, Paul J; Watson, Jonathan M; Barbastathis, George; Barton, Jennifer K; Kostuk, Raymond K

    2008-09-15

    A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously.

  1. Liquid-crystal tunable filter spectral imaging for brain tumor demarcation

    NASA Astrophysics Data System (ADS)

    Gebhart, Steven C.; Thompson, Reid C.; Mahadevan-Jansen, Anita

    2007-04-01

    Past studies have demonstrated that combined fluorescence and diffuse reflectance spectroscopy can successfully discriminate between normal, tumor core, and tumor margin tissues in the brain. To achieve efficient, real-time surgical resection guidance with optical biopsy, probe-based spectroscopy must be extended to spectral imaging to spatially demarcate the tumor margins. We describe the design and characterization of a combined fluorescence and diffuse reflectance imaging system that uses liquid-crystal tunable filter technology. Experiments were conducted to quantitatively determine the linearity, field of view, spatial and spectral resolution, and wavelength sensitivity of the imaging system. Spectral images were acquired from tissue phantoms, mouse brain in vitro, and human cortex in vivo for functional testing of the system. The spectral imaging system produces measured intensities that are linear with sample emission intensity and integration time and possesses a 1 in. (2.54 cm) field of view for a 7 in. (18 cm) object distance. The spectral resolution is linear with wavelength, and the spatial resolution is pixel-limited. The sensitivity spectra for the imaging system provide a guide for the distribution of total image integration time between wavelengths. Functional tests in vitro demonstrate the capability to spectrally discriminate between brain tissues based on exogenous fluorescence contrast or endogenous tissue composition. In vivo imaging captures adequate fluorescence and diffuse reflectance intensities within a clinically viable 2 min imaging time frame and demonstrates the importance of hemostasis to acquired signal strengths and imaging speed.

  2. [Design of full-polarized and multi-spectral imaging system based on LCVR].

    PubMed

    Zhang, Ying; Zhao, Hui-jie; Cheng, Xuan; Xiong, Sheng-jun

    2011-05-01

    A new full-polarized multi-spectral imaging system is described, which uses electronically controlled LCVR (liquid crystal variable retarder) to modulate the full-polarized state of light in the visible to IR range. The system consisted of optical lenses, LCVRs, filters and CCD. Firstly, the system structure, working theory and optical design are introduced in the present paper. A polarization calibration method is provided and the calibration system was set up, which realized high-precision polarization calibration using a small polarized source. Then, a field experiment with the imaging system was carried out. Polarized spectral images with higher spectral and spatial resolution were collected. Finally, the data acquired were rough processed to get polarization degree image of the targets. It is concluded that the experiment has proved that the imaging system is effective in obtaining full-polarized and multi- spectral data. The image captured by the system can be applied to object identification and object classification.

  3. Airborne hyperspectral imaging in the visible-to-mid wave infrared spectral range by fusing three spectral sensors

    NASA Astrophysics Data System (ADS)

    Jakovels, Dainis; Filipovs, Jevgenijs; Erinš, Gatis; Taskovs, Juris

    2014-10-01

    Airborne hyperspectral imaging is widely used for remote sensing of environment. The choice of spectral region usually depends on the availability and cost of the sensor. Visible-to-near infrared (400-1100 nm) spectral range corresponds to spectral sensitivity of relatively cheap Si detectors therefore it is the most commonly used. The implementation of shortwave infrared (1100-3000 nm) requires more expensive solutions, but can provide valuable information about the composition of the substance. Mid wave infrared (3000-8000 nm) is rarely used for civilian applications, but it provides information on the thermal emission of materials. The fusion of different sensors allows spectral analysis of a wider spectral range combining and improving already existing algorithms for the analysis of chemical content and classification. Here we introduce our Airborne Surveillance and Environmental Monitoring System (ARSENAL) that was developed by fusing seven sensors. The first test results from the fusion of three hyperspectral imaging sensors in the visible-to-mid wave infrared (365-5000 nm) are demonstrated. Principal component analysis (PCA) is applied to test correlation between principal components (PCs) and common vegetation indices.

  4. Point spread function engineering with multiphoton SPIFI

    NASA Astrophysics Data System (ADS)

    Wernsing, Keith A.; Field, Jeffrey J.; Domingue, Scott R.; Allende-Motz, Alyssa M.; DeLuca, Keith F.; Levi, Dean H.; DeLuca, Jennifer G.; Young, Michael D.; Squier, Jeff A.; Bartels, Randy A.

    2016-03-01

    MultiPhoton SPatIal Frequency modulated Imaging (MP-SPIFI) has recently demonstrated the ability to simultaneously obtain super-resolved images in both coherent and incoherent scattering processes -- namely, second harmonic generation and two-photon fluorescence, respectively.1 In our previous analysis, we considered image formation produced by the zero and first diffracted orders from the SPIFI modulator. However, the modulator is a binary amplitude mask, and therefore produces multiple diffracted orders. In this work, we extend our analysis to image formation in the presence of higher diffracted orders. We find that tuning the mask duty cycle offers a measure of control over the shape of super-resolved point spread functions in an MP-SPIFI microscope.

  5. Multiphoton microscopy of cleared mouse organs

    NASA Astrophysics Data System (ADS)

    Parra, Sonia G.; Chia, Thomas H.; Zinter, Joseph P.; Levene, Michael J.

    2010-05-01

    Typical imaging depths with multiphoton microscopy (MPM) are limited to less than 300 μm in many tissues due to light scattering. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having a similar index of refraction to that of proteins. We demonstrate MPM of intact, fixed, cleared mouse organs with penetration depths and fields of view in excess of 2 mm. MPM enables the creation of large 3-D data sets with flexibility in pixel format and ready access to intrinsic fluorescence and second-harmonic generation. We present high-resolution images and 3-D image stacks of the brain, small intestine, large intestine, kidney, lung, and testicle with image sizes as large as 4096×4096 pixels.

  6. SAR image segmentation with entropy ranking based adaptive semi-supervised spectral clustering

    NASA Astrophysics Data System (ADS)

    Zhang, Xiangrong; Yang, Jie; Hou, Biao; Jiao, Licheng

    2010-10-01

    Spectral clustering has become one of the most popular modern clustering algorithms in recent years. In this paper, a new algorithm named entropy ranking based adaptive semi-supervised spectral clustering for SAR image segmentation is proposed. We focus not only on finding a suitable scaling parameter but also determining automatically the cluster number with the entropy ranking theory. Also, two kinds of constrains must-link and cannot-link based semi-supervised spectral clustering is applied to gain better segmentation results. Experimental results on SAR images show that the proposed method outperforms other spectral clustering algorithms.

  7. Multiphoton tomography of astronauts

    NASA Astrophysics Data System (ADS)

    König, Karsten; Weinigel, Martin; Pietruszka, Anna; Bückle, Rainer; Gerlach, Nicole; Heinrich, Ulrike

    2015-03-01

    Weightlessness may impair the astronaut's health conditions. Skin impairments belong to the most frequent health problems during space missions. Within the Skin B project, skin physiological changes during long duration space flights are currently investigated on three European astronauts that work for nearly half a year at the ISS. Measurements on the hydration, the transepidermal water loss, the surface structure, elasticity and the tissue density by ultrasound are conducted. Furthermore, high-resolution in vivo histology is performed by multiphoton tomography with 300 nm spatial and 200 ps temporal resolution. The mobile certified medical tomograph with a flexible 360° scan head attached to a mechano-optical arm is employed to measure two-photon autofluorescence and SHG in the volar forearm of the astronauts. Modification of the tissue architecture and of the fluorescent biomolecules NAD(P)H, keratin, melanin and elastin are detected as well as of SHG-active collagen. Thinning of the vital epidermis, a decrease of the autofluoresence intensity, an increase in the long fluorescence lifetime, and a reduced skin ageing index SAAID based on an increased collagen level in the upper dermis have been found. Current studies focus on recovery effects.

  8. Multiphoton Effects in Rutile.

    NASA Astrophysics Data System (ADS)

    Royce, Gerald A.

    Multiphoton effects are investigated in crystalline rutile TiO(,2) using Nd:YAG laser photons. The 1.06 micron laser is operated in Q-switched mode with intensities up to 1.4 x 10('6) watts/cm('2) on the rutile crystal. Photoconductivity measurements provide data indicating a mixture of modes for electrons to be photoionized. Assuming aluminum impurity as the contributing sites, the first order photionization cross section is found to be 1.5 x 10('-26) cm('2) and second order cross section is found to be 7.7 x 10('-51) cm('4)-s. No appreciable change in cross section is observed for circular versus linear polarization of the laser. Observations of the photo-emission of the laser illuminated crystal provide radiative relaxation times on the order of 100 nanoseconds with emission peaks at 4500 and 5000 angstroms plus a near infrared continuum out to 1 micron. The thermoluminescence of rutile shows a number of trapping levels between 0.4 and 0.8 eV below the conduction band. These are attributed to an aluminum impurity.

  9. [Influence of human body target's spectral characteristics on visual range of low light level image intensifiers].

    PubMed

    Zhang, Jun-Ju; Yang, Wen-Bin; Xu, Hui; Liu, Lei; Tao, Yuan-Yaun

    2013-11-01

    To study the effect of different human target's spectral reflective characteristic on low light level (LLL) image intensifier's distance, based on the spectral characteristics of the night-sky radiation and the spectral reflective coefficients of common clothes, we established a equation of human body target's spectral reflective distribution, and analyzed the spectral reflective characteristics of different human targets wearing the clothes of different color and different material, and from the actual detection equation of LLL image intensifier distance, discussed the detection capability of LLL image intensifier for different human target. The study shows that the effect of different human target's spectral reflective characteristic on LLL image intensifier distance is mainly reflected in the average reflectivity rho(-) and the initial contrast of the target and the background C0. Reflective coefficient and spectral reflection intensity of cotton clothes are higher than polyester clothes, and detection capability of LLL image intensifier is stronger for the human target wearing cotton clothes. Experimental results show that the LLL image intensifiers have longer visual ranges for targets who wear cotton clothes than targets who wear same color but polyester clothes, and have longer visual ranges for targets who wear light-colored clothes than targets who wear dark-colored clothes. And in the full moon illumination conditions, LLL image intensifiers are more sensitive to the clothes' material.

  10. Compact multispectral fluorescence imaging system with spectral multiplexed volume holographic grating

    NASA Astrophysics Data System (ADS)

    Lv, Yanlu; Cai, Chuangjian; Bai, Jing; Luo, Jianwen

    2016-12-01

    Traditional spectral imaging systems mainly rely on spatial scanning or spectral scanning methods to acquire spatial and spectral features. The acquisition is time-consuming and cannot fully satisfy the need of monitoring dynamic phenomenon and observing different structures of the specimen simultaneously. To overcome these barriers, we develop a video-rate simultaneous multispectral imaging system built with a spectral multiplexed volume holographic grating (VHG) and few optical components. Four spectral multiplexed volume holograms optimized for four discrete spectral bands (centered at 488 nm, 530 nm, 590 nm and 620 nm) are recorded into an 8×12 mm photo-thermal refractive glass. The diffraction efficiencies of all the holograms within the multiplexed VHG are greater than 80%. With the high throughout multiplexed VHG, the system can work with both reflection and fluorescence modes and allow simultaneous acquisition of spectral and spatial information with a single exposure. Imaging experiments demonstrate that the multispectral images of the target illuminated with white light source can be obtained. Fluorescence images of multiple fluorescence objects (two glass beads filled with 20 uL 1.0 mg/mL quantum dots solutions that emit 530 +/- 15 nm and 620 +/- 15 nm fluorescence, respectively) buried 3 mm below the surface of a tissue mimicking phantom are acquired. The results demonstrate that the system can provide complementary information in fluorescence imaging. The design diagram of the proposed system is given to explain the advantage of compactness and flexibility in integrating with other imaging platforms.

  11. Contrast enhancement of subcutaneous blood vessel images by means of visible and near-infrared hyper-spectral imaging

    NASA Astrophysics Data System (ADS)

    Katrašnik, Jaka; Bürmen, Miran; Pernuš, Franjo; Likar, Boštjan

    2009-02-01

    Visualization of subcutaneous veins is very difficult with the naked eye, but important for diagnosis of medical conditions and different medical procedures such as catheter insertion and blood withdrawal. Moreover, recent studies showed that the images of subcutaneous veins could be used for biometric identification. The majority of methods used for enhancing the contrast between the subcutaneous veins and surrounding tissue are based on simple imaging systems utilizing CMOS or CCD cameras with LED illumination capable of acquiring images from the near infrared spectral region, usually near 900 nm. However, such simplified imaging methods cannot exploit the full potential of the spectral information. In this paper, a new highly versatile method for enhancing the contrast of subcutaneous veins based on state-of-the-art high-resolution hyper-spectral imaging system utilizing the spectral region from 550 to 1700 nm is presented. First, a detailed analysis of the contrast between the subcutaneous veins and the surrounding tissue as a function of wavelength, for several different positions on the human arm, was performed in order to extract the spectral regions with the highest contrast. The highest contrast images were acquired at 1100 nm, however, combining the individual images from the extracted spectral regions by the proposed contrast enhancement method resulted in a single image with up to ten-fold better contrast. Therefore, the proposed method has proved to be a useful tool for visualization of subcutaneous veins.

  12. Comparison of local and global angular interpolation applied to spectral-spatial EPR image reconstruction.

    PubMed

    Ahn, Kang-Hyun; Halpern, Howard J

    2007-03-01

    Spectral-spatial images reconstructed from a small number of projections suffer from streak artifacts that are seen as noise, particularly in the spectral dimension. Interpolation in projection space can reduce artifacts in the reconstructed images. The reduction of background artifacts improves lineshape fitting. In this work, we compared the performances of angular interpolation implemented using linear, cubic B-spline, and sinc methods. Line width maps were extracted from 4-D EPR images of phantoms using spectral fitting to evaluate each interpolation method and its robustness to noise. Results from experiment and simulation showed that the cubic B-spline, angular interpolation was preferable to either sinc or linear interpolation methods.

  13. Initial clinical testing of a multi-spectral imaging system built on a smartphone platform

    NASA Astrophysics Data System (ADS)

    Mink, Jonah W.; Wexler, Shraga; Bolton, Frank J.; Hummel, Charles; Kahn, Bruce S.; Levitz, David

    2016-03-01

    Multi-spectral imaging systems are often expensive and bulky. An innovative multi-spectral imaging system was fitted onto a mobile colposcope, an imaging system built around a smartphone in order to image the uterine cervix from outside the body. The multi-spectral mobile colposcope (MSMC) acquires images at different wavelengths. This paper presents the clinical testing of MSMC imaging (technical validation of the MSMC system is described elsewhere 1 ). Patients who were referred to colposcopy following abnormal screening test (Pap or HPV DNA test) according to the standard of care were enrolled. Multi-spectral image sets of the cervix were acquired, consisting of images from the various wavelengths. Image acquisition took 1-2 sec. Areas suspected for dysplasia under white light imaging were biopsied, according to the standard of care. Biopsied sites were recorded on a clockface map of the cervix. Following the procedure, MSMC data was processed from the sites of biopsied sites. To date, the initial histopathological results are still outstanding. Qualitatively, structures in the cervical images were sharper at lower wavelengths than higher wavelengths. Patients tolerated imaging well. The result suggests MSMC holds promise for cervical imaging.

  14. Multi-spectral image enhancement algorithm based on keeping original gray level

    NASA Astrophysics Data System (ADS)

    Wang, Tian; Xu, Linli; Yang, Weiping

    2016-11-01

    Characteristics of multi-spectral imaging system and the image enhancement algorithm are introduced.Because histogram equalization and some other image enhancement will change the original gray level,a new image enhancement algorithm is proposed to maintain the gray level.For this paper, we have chosen 6 narrow-bands multi-spectral images to compare,the experimental results show that the proposed method is better than those histogram equalization and other algorithm to multi-spectral images.It also insures that histogram information contained in original features is preserved and guarantees to make use of data class information.What's more,on the combination of subjective and objective sharpness evaluation,details of the images are enhanced and noise is weaken.

  15. Design and fabrication of Fourier spectral filter array for multispectral imaging

    NASA Astrophysics Data System (ADS)

    Ni, Chuan; Jia, Jie; Hirakawa, Keigo; Sarangan, Andrew

    2016-09-01

    Multispectral imaging has the capability to identify the state of objects based on their spectral characteristics. These are features not available with conventional color imaging based on metameric RGB (red, green and blue) colors alone. Current multispectral imaging systems use narrowband filters to capture the spectral content of a scene, which necessitates different filters to be designed and applied for each application. Previously, we demonstrated the concept of Fourier multispectral imaging using filters with sinusoidally varying transmittance [1, 2]. In this paper, we report to the design of a five channel, spatially multiplexed pixel filter array. This enables single-shot images and makes it possible to capture scenes containing moving objects.

  16. Color image segmentation using watershed and Nyström method based spectral clustering

    NASA Astrophysics Data System (ADS)

    Bai, Xiaodong; Cao, Zhiguo; Yu, Zhenghong; Zhu, Hu

    2011-11-01

    Color image segmentation draws a lot of attention recently. In order to improve efficiency of spectral clustering in color image segmentation, a novel two-stage color image segmentation method is proposed. In the first stage, we use vector gradient approach to detect color image gradient information, and watershed transformation to get the pre-segmentation result. In the second stage, Nyström extension based spectral clustering is used to get the final result. To verify the proposed algorithm, it is applied to color images from the Berkeley Segmentation Dataset. Experiments show our method can bring promising results and reduce the runtime significantly.

  17. Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags.

    PubMed

    Hackl, Matthias J; Burford, James L; Villanueva, Karie; Lam, Lisa; Suszták, Katalin; Schermer, Bernhard; Benzing, Thomas; Peti-Peterdi, János

    2013-12-01

    Podocytes are critical in the maintenance of a healthy glomerular filter; however, they have been difficult to study in the intact kidney because of technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PECs) in vivo. In podocin-GFP mice, podocytes formed sporadic multicellular clusters after unilateral ureteral ligation and migrated into the parietal Bowman's capsule. The tracking of single cells in podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP or RFP revealed the simultaneous migration of multiple podocytes. In phosphoenolpyruvate carboxykinase (PEPCK)-GFP mice, serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support a highly dynamic rather than a static nature of the glomerular environment and cellular composition. Future application of this new approach should advance our understanding of the mechanisms of glomerular injury and regeneration.

  18. Rapid mesoscale multiphoton microscopy of human skin

    PubMed Central

    Balu, Mihaela; Mikami, Hideharu; Hou, Jue; Potma, Eric O.; Tromberg, Bruce J.

    2016-01-01

    We present a multiphoton microscope designed for mesoscale imaging of human skin. The system is based on two-photon excited fluorescence and second-harmonic generation, and images areas of ~0.8x0.8 mm2 at speeds of 0.8 fps (800x800 pixels; 12 frame averages) for high signal-to-noise ratio, with lateral and axial resolutions of 0.5µm and 3.3µm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, optimized relay optics, a beam expander and high NA objective lens. Computed aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide areas in normal human skin. PMID:27895980

  19. Validation of spectral sky radiance derived from all-sky camera images - a case study

    NASA Astrophysics Data System (ADS)

    Tohsing, K.; Schrempf, M.; Riechelmann, S.; Seckmeyer, G.

    2014-01-01

    Spectral sky radiance (380-760 nm) is derived from measurements with a Hemispherical Sky Imager (HSI) system. The HSI consists of a commercial compact CCD (charge coupled device) camera equipped with a fish-eye lens and provides hemispherical sky images in three reference bands such as red, green and blue. To obtain the spectral sky radiance from these images non-linear regression functions for various sky conditions have been derived. The camera-based spectral sky radiance was validated by spectral sky radiance measured with a CCD spectroradiometer. The spectral sky radiance for complete distribution over the hemisphere between both instruments deviates by less than 20% at 500 nm for all sky conditions and for zenith angles less than 80°. The reconstructed spectra of the wavelength 380 nm to 760 nm between both instruments at various directions deviate by less then 20% for all sky conditions.

  20. Validation of spectral sky radiance derived from all-sky camera images - a case study

    NASA Astrophysics Data System (ADS)

    Tohsing, K.; Schrempf, M.; Riechelmann, S.; Seckmeyer, G.

    2014-07-01

    Spectral sky radiance (380-760 nm) is derived from measurements with a hemispherical sky imager (HSI) system. The HSI consists of a commercial compact CCD (charge coupled device) camera equipped with a fish-eye lens and provides hemispherical sky images in three reference bands such as red, green and blue. To obtain the spectral sky radiance from these images, non-linear regression functions for various sky conditions have been derived. The camera-based spectral sky radiance was validated using spectral sky radiance measured with a CCD spectroradiometer. The spectral sky radiance for complete distribution over the hemisphere between both instruments deviates by less than 20% at 500 nm for all sky conditions and for zenith angles less than 80°. The reconstructed spectra of the wavelengths 380-760 nm between both instruments at various directions deviate by less than 20% for all sky conditions.

  1. New confocal microscopy hyperspectral imager for NIR-emitting bioprobes: high spectral resolution for a wide spectral range (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Marcet, Stéphane; Benayas, Antonio; Quintanilla, Marta; Mangiarini, Francesca; Verhaegen, Marc; Vetrone, Fiorenzo; Blais-Ouellette, Sébastien

    2016-03-01

    Functional nanoscale materials are being extensively investigated for applications in biology and medicine and are ready to make significant contributions in the realization of exciting advancements in diverse areas of diagnostics and therapeutics. Aiming for more accurate, efficient, non-invasive and fast diagnostic tools, the use of near-infrared (NIR) light in the range of the 1st and 2nd biological window (NIR-I: 0.70-0.95 µm; NIR-II: 1.00-1.35 µm) provides deeper penetration depth into biological tissue, better image contrast, reduced phototoxicity and photobleaching. Consequently, NIR-based bioimaging became a quickly emerging field and manifold new NIR-emitting bioprobes have been reported. Since commercially available microscopes are not optimized for this kind of NPs, a new microscopy hyperspectral confocal imager has been developed to cover a broad spectral range (400 to 1700 nm) with high spectral resolution. The smallest spectral variation can be easily monitored thanks to the high spectral resolution (as low as 0.2 nm). This is possible thanks to a combination of an EMCCD and an InGaAs camera with a high resolution spectrometer. An extended number of NPs can be excited with a Ti:Sapphire laser, which provides tunable illumination within 690-1040 nm. Cells and tissues can be mapped in less than 100 ms, allowing in-vivo imaging. As a proof of concept, here we present the preliminary results of the spatial distribution of the fluorescence signal intensity from lanthanide doped nanoparticles incorporated into a system of biological interest. The temperature sub-mm gradient - analyzing the spectral features so gathered through an all-optical route is also thoroughly discussed.

  2. Spectral CT imaging of vulnerable plaque with two independent biomarkers

    NASA Astrophysics Data System (ADS)

    Baturin, Pavlo; Alivov, Yahya; Molloi, Sabee

    2012-07-01

    The purpose of this paper is to investigate the feasibility of a novel four-material decomposition technique for assessing the vulnerability of plaque with two contrast materials spectral computer tomography (CT) using two independent markers: plaque's inflammation and spotty calcification. A simulation study was conducted using an energy-sensitive photon-counting detector for k-edge imaging of the coronary arteries. In addition to detecting the inflammation status, which is known as a biological marker of a plaque's vulnerability, we use spotty calcium concentration as an independent marker to test a plaque's vulnerability. We have introduced a new method for detecting and quantifying calcium concentrations in the presence of two contrast materials (iodine and gold), calcium and soft tissue background. In this method, four-material decomposition was performed on a pixel-by-pixel basis, assuming there was an arbitrary mixture of materials in the voxel. The concentrations of iodine and gold were determined by the k-edge material decomposition based on the maximum likelihood method. The calibration curves of the attenuation coefficients, with respect to the concentrations of different materials, were used to separate the calcium signal from both contrast materials and different soft tissues in the mixtures. Three different materials (muscle, blood and lipid) were independently used as soft tissue. The simulations included both ideal and more realistic energy resolving detectors to measure the polychromatic photon spectrum in single slice parallel beam geometry. The ideal detector was used together with a 3 cm diameter digital phantom to demonstrate the decomposition method while a more realistic detector and a 33 × 24 cm2 digital chest phantom were simulated to validate the vulnerability assessment technique. A 120 kVp spectrum was generated to produce photon flux sufficient for detecting contrast materials above the k-edges of iodine (33.2 keV) and gold (80.7 ke

  3. TICMR: Total Image Constrained Material Reconstruction via Nonlocal Total Variation Regularization for Spectral CT.

    PubMed

    Liu, Jiulong; Ding, Huanjun; Molloi, Sabee; Zhang, Xiaoqun; Gao, Hao

    2016-12-01

    This work develops a material reconstruction method for spectral CT, namely Total Image Constrained Material Reconstruction (TICMR), to maximize the utility of projection data in terms of both spectral information and high signal-to-noise ratio (SNR). This is motivated by the following fact: when viewed as a spectrally-integrated measurement, the projection data can be used to reconstruct a total image without spectral information, which however has a relatively high SNR; when viewed as a spectrally-resolved measurement, the projection data can be utilized to reconstruct the material composition, which however has a relatively low SNR. The material reconstruction synergizes material decomposition and image reconstruction, i.e., the direct reconstruction of material compositions instead of a two-step procedure that first reconstructs images and then decomposes images. For material reconstruction with high SNR, we propose TICMR with nonlocal total variation (NLTV) regularization. That is, first we reconstruct a total image using spectrally-integrated measurement without spectral binning, and build the NLTV weights from this image that characterize nonlocal image features; then the NLTV weights are incorporated into a NLTV-based iterative material reconstruction scheme using spectrally-binned projection data, so that these weights serve as a high-SNR reference to regularize material reconstruction. Note that the nonlocal property of NLTV is essential for material reconstruction, since material compositions may have significant local intensity variations although their structural information is often similar. In terms of solution algorithm, TICMR is formulated as an iterative reconstruction method with the NLTV regularization, in which the nonlocal divergence is utilized based on the adjoint relationship. The alternating direction method of multipliers is developed to solve this sparsity optimization problem. The proposed TICMR method was validated using both simulated

  4. Brain tumor demarcation with liquid-crystal tunable filter spectral imaging

    NASA Astrophysics Data System (ADS)

    Gebhart, Steven C.; Mahadevan-Jansen, Anita

    2006-02-01

    Past studies have demonstrated that combined fluorescence and diffuse reflectance spectroscopy can successfully discriminate between normal, tumor core, and tumor margin tissues in the brain. To achieve efficient surgical resection guidance with optical biopsy, probe-based spectroscopy must be extended to spectral imaging to spatially demarcate the tumor margins. This paper describes the design and testing of a combined fluorescence and diffuse reflectance imaging system which uses liquid-crystal tunable filter technology. Experiments were conducted to quantitatively determine its linearity, field of view, spatial and spectral resolution, and wavelength sensitivity. For functional testing, spectral images were acquired from tissue phantoms, mouse brain in vitro, and rat brain cortex in vivo. The spectral imaging system is characterized by measured intensities which are linear with sample emission intensity and integration time, a one-inch field of view for a seven-inch object distance, spectral resolution which is linear with wavelength, spatial resolution which is pixel-limited, and sensitivity functions which provide a guide for the distribution of total image integration time between wavelengths. Functional testing demonstrated good spatial and spectral constrast between brain tissue types, the capability to acquire adequate fluorescence and diffuse reflectance intensities within a one-minute imaging timeframe, and the importance of hemostasis to acquired signal strengths and imaging speed.

  5. Improving signal levels in intravital multiphoton microscopy using an objective correction collar

    NASA Astrophysics Data System (ADS)

    Muriello, Pamela A.; Dunn, Kenneth W.

    2008-04-01

    Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point-spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreases the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.

  6. Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells.

    PubMed

    Zhang, Lili; Qin, Guiqi; Chai, Liuying; Zhang, Jiang; Yang, Fangfang; Yang, Hongqin; Xie, Shusen; Chen, Tongsheng

    2015-08-01

    With its precise, sensitive, and nondestructive features, spectral unmixing-based fluorescence resonance energy transfer (FRET) microscopy has been widely applied to visualize intracellular biological events. In this report, we set up a spectral wide-field microscopic FRET imaging system by integrating a varispec liquid crystal tunable filter into a wide-field microscope for quantitative FRET measurement in living cells. We implemented a representative emission-spectral unmixing-based FRET measurement method on this platform to simultaneously acquire pixel-to-pixel images of both FRET efficiency (E ) and acceptor-to-donor concentration ratio (R C ) in living HepG2 cells expressing fusion proteins in the presence or absence of free donors and acceptors and obtained consistent results with other instruments and methods. This stable and low-cost spectral wide-field microscopic FRET imaging system provides a new toolbox for imaging molecular events with high spatial resolution in living cells.

  7. RELIABILITY OF CONFOCAL MICROSCOPY SPECTRAL IMAGING SYSTEMS: USE OF MULTISPECTRAL BEADS

    EPA Science Inventory

    Background: There is a need for a standardized, impartial calibration, and validation protocol on confocal spectral imaging (CSI) microscope systems. To achieve this goal, it is necessary to have testing tools to provide a reproducible way to evaluate instrument performance. ...

  8. Intelligent spectral signature bio-imaging in vivo for surgical applications

    NASA Astrophysics Data System (ADS)

    Jeong, Jihoon; Frykman, Philip K.; Gaon, Mark; Chung, Alice P.; Lindsley, Erik H.; Hwang, Jae Y.; Farkas, Daniel L.

    2007-02-01

    Multi-spectral imaging provides digital images of a scene or object at a large, usually sequential number of wavelengths, generating precise optical spectra at every pixel. We use the term "spectral signature" for a quantitative plot of optical property variations as a function of wavelengths. We present here intelligent spectral signature bio-imaging methods we developed, including automatic signature selection based on machine learning algorithms and database search-based automatic color allocations, and selected visualization schemes matching these approaches. Using this intelligent spectral signature bio-imaging method, we could discriminate normal and aganglionic colon tissue of the Hirschsprung's disease mouse model with over 95% sensitivity and specificity in various similarity measure methods and various anatomic organs such as parathyroid gland, thyroid gland and pre-tracheal fat in dissected neck of the rat in vivo.

  9. Optical imaging of hemoglobin oxygen saturation using a small number of spectral images for endoscopic application

    NASA Astrophysics Data System (ADS)

    Saito, Takaaki; Yamaguchi, Hiroshi

    2015-12-01

    Tissue hypoxia is associated with tumor and inflammatory diseases, and detection of hypoxia is potentially useful for their detailed diagnosis. An endoscope system that can optically observe hemoglobin oxygen saturation (StO2) would enable minimally invasive, real-time detection of lesion hypoxia in vivo. Currently, point measurement of tissue StO2 via endoscopy is possible using the commercial fiber-optic oximeter T-Stat, which is based on visible light spectroscopy at many wavelengths. For clinical use, however, imaging of StO2 is desirable to assess the distribution of tissue oxygenation around a lesion. Here, we describe our StO2 imaging technique based on a small number of wavelength ranges in the visible range. By assuming a homogeneous tissue, we demonstrated that tissue StO2 can be obtained independently from the scattering property and blood concentration of tissue using four spectral bands. We developed a prototype endoscope system and used it to observe tissue-simulating phantoms. The StO2 (%) values obtained using our technique agreed with those from the T-Stat within 10%. We also showed that tissue StO2 can be derived using three spectral band if the scattering property is fixed at preliminarily measured values.

  10. Multiscale target extraction using a spectral saliency map for a hyperspectral image.

    PubMed

    Zhang, Jing; Geng, Wenhao; Zhuo, Li; Tian, Qi; Cao, Yan

    2016-10-01

    With the rapid growth of the capabilities for hyperspectral imagery acquisition, how to efficiently find the significant target in hyperspectral imagery has become a fundamental task for remote-sensing applications. Existing target extraction methods mainly separate targets from background with a threshold based on pixels and single-scale image information extraction. However, due to the high dimensional characteristics and the complex background of hyperspectral imagery, it is difficult to obtain good extraction results with existing methods. Saliency detection has been a promising topic because saliency features can quickly locate saliency regions from complex backgrounds. Considering the spatial and spectral characteristics of a hyperspectral image, a multiscale target extraction method using a spectral saliency map is proposed for a hyperspectral image, which includes: (1) a spectral saliency model is constructed for detecting spectral saliency map in a hyperspectral image; (2) focus of attention (FOA) as the seed point is competed in the spectral saliency map by the winner-take-all (WTA) network; (3) the multiscale image is segmented by region growing based on the minimum-heterogeneity rule after calculating the heterogeneity of the seed point with its surrounding pixels; (4) the salient target is detected and segmented under the constraint of the spectral saliency map. The experimental results show that the proposed method can effectively improve the accuracy of target extraction for hyperspectral images.

  11. Subsurface imaging using the spectral polarization difference technique and NIR illumination

    SciTech Connect

    Alfano, R R; Demos, S G; Radousky, H B

    1999-01-26

    A subsurface imaging system is utilized to test the ability of the spectral polarization difference imaging technique for deep subsurface imaging in tissues. The illumination of the system is derived from compact class III lasers in the red and NIR spectral region and, alternatively, from a white light source and selection of the appropriate illumination wavelength using band-pass optical filters. The experimental results demonstrate detection and imaging of a high-scattering object located up to 1.5 cm underneath the surface of a host chicken tissue.

  12. Sensitivity study on the spectral calibration of a hyper-spectral imaging spectrometer for the GEO-KOMPSAT2

    NASA Astrophysics Data System (ADS)

    Kang, M.; Ahn, M.

    2013-12-01

    The next generation of geostationary earth observing satellite program of Korea (GEO-KOMPSAT-2A&B) is under development. While the GEO-KOMPSAT-2A is dedicated for the operational weather mission and planed to be launched in 2017, the second one will have ocean and environmental mission with planed launch of 2018. For the environmental mission, a hyperspectral spectrometer named the Global Environment Measuring Spectrometer (GEMS) designed to monitor the important trace gases such as O3, SO2, NO2, HCHO and aerosols which affect directly and indirectly the air quality will be onboard the second satellite with a ocean color imager. Based on the preliminary design concept, the GEMS instrument utilizes a reflecting telescope with the Offner spectrometer which uses the grating and 2D CCD (1 for spatial and another for spectral). Due to the nature of instrumentations, there is always possibility of wavelength shift and squeeze at the measured raw radiance from the CCD. Thus, it is important to have a proper algorithm for the accurate spectral calibration. Currently, we plan to have a two-step process for an accurate spectral calibration. First step is done by the application of spectral calibration process provided by instrument manufacturer which will be applied to whole observation wavelength band. The second step which will be applied for each wavelength bands used for the retrieval will be using the high resolution solar spectrum for the reference spectrum used for fitting the measured radiances and irradiances. For the application of second step, there are several important pre-requisite information which could be obtained through the ground test of the instrument or through the actual measurement data or through assumptions. Here we investigate the sensitivity of the spectral calibration accuracy to the important parameters such as the spectral response function of each band, band width, undersampling correction, and so on, The simulated sensitivity tests will be

  13. Spectral compression algorithms for the analysis of very large multivariate images

    DOEpatents

    Keenan, Michael R.

    2007-10-16

    A method for spectrally compressing data sets enables the efficient analysis of very large multivariate images. The spectral compression algorithm uses a factored representation of the data that can be obtained from Principal Components Analysis or other factorization technique. Furthermore, a block algorithm can be used for performing common operations more efficiently. An image analysis can be performed on the factored representation of the data, using only the most significant factors. The spectral compression algorithm can be combined with a spatial compression algorithm to provide further computational efficiencies.

  14. Stimulated Raman hyperspectral imaging based on spectral filtering of broadband fiber laser pulses.

    PubMed

    Ozeki, Yasuyuki; Umemura, Wataru; Sumimura, Kazuhiko; Nishizawa, Norihiko; Fukui, Kiichi; Itoh, Kazuyoshi

    2012-02-01

    We demonstrate a technique of hyperspectral imaging in stimulated Raman scattering (SRS) microscopy using a tunable optical filter, whose transmission wavelength can be varied quickly by a galvanometer mirror. Experimentally, broadband Yb fiber laser pulses are synchronized with picosecond Ti:sapphire pulses, and then spectrally filtered out by the filter. After amplification by fiber amplifiers, we obtain narrowband pulses with a spectral width of <3.3 cm(-1) and a wavelength tunability of >225 cm(-1). By using these pulses, we accomplish SRS imaging of polymer beads with spectral information.

  15. Spectral feature variations in x-ray diffraction imaging systems

    NASA Astrophysics Data System (ADS)

    Wolter, Scott D.; Greenberg, Joel A.

    2016-05-01

    Materials with different atomic or molecular structures give rise to unique scatter spectra when measured by X-ray diffraction. The details of these spectra, though, can vary based on both intrinsic (e.g., degree of crystallinity or doping) and extrinsic (e.g., pressure or temperature) conditions. While this sensitivity is useful for detailed characterizations of the material properties, these dependences make it difficult to perform more general classification tasks, such as explosives threat detection in aviation security. A number of challenges, therefore, currently exist for reliable substance detection including the similarity in spectral features among some categories of materials combined with spectral feature variations from materials processing and environmental factors. These factors complicate the creation of a material dictionary and the implementation of conventional classification and detection algorithms. Herein, we report on two prominent factors that lead to variations in spectral features: crystalline texture and temperature variations. Spectral feature comparisons between materials categories will be described for solid metallic sheet, aqueous liquids, polymer sheet, and metallic, organic, and inorganic powder specimens. While liquids are largely immune to texture effects, they are susceptible to temperature changes that can modify their density or produce phase changes. We will describe in situ temperature-dependent measurement of aqueous-based commercial goods in the temperature range of -20°C to 35°C.

  16. Stepwise method based on Wiener estimation for spectral reconstruction in spectroscopic Raman imaging.

    PubMed

    Chen, Shuo; Wang, Gang; Cui, Xiaoyu; Liu, Quan

    2017-01-23

    Raman spectroscopy has demonstrated great potential in biomedical applications. However, spectroscopic Raman imaging is limited in the investigation of fast changing phenomena because of slow data acquisition. Our previous studies have indicated that spectroscopic Raman imaging can be significantly sped up using the approach of narrow-band imaging followed by spectral reconstruction. A multi-channel system was built to demonstrate the feasibility of fast wide-field spectroscopic Raman imaging using the approach of simultaneous narrow-band image acquisition followed by spectral reconstruction based on Wiener estimation in phantoms. To further improve the accuracy of reconstructed Raman spectra, we propose a stepwise spectral reconstruction method in this study, which can be combined with the earlier developed sequential weighted Wiener estimation to improve spectral reconstruction accuracy. The stepwise spectral reconstruction method first reconstructs the fluorescence background spectrum from narrow-band measurements and then the pure Raman narrow-band measurements can be estimated by subtracting the estimated fluorescence background from the overall narrow-band measurements. Thereafter, the pure Raman spectrum can be reconstructed from the estimated pure Raman narrow-band measurements. The result indicates that the stepwise spectral reconstruction method can improve spectral reconstruction accuracy significantly when combined with sequential weighted Wiener estimation, compared with the traditional Wiener estimation. In addition, qualitatively accurate cell Raman spectra were successfully reconstructed using the stepwise spectral reconstruction method from the narrow-band measurements acquired by a four-channel wide-field Raman spectroscopic imaging system. This method can potentially facilitate the adoption of spectroscopic Raman imaging to the investigation of fast changing phenomena.

  17. Visible spectral imager for occultation and nightglow (VISION) for the PICASSO Mission

    NASA Astrophysics Data System (ADS)

    Saari, Heikki; Näsilä, Antti; Holmlund, Christer; Mannila, Rami; Näkki, Ismo; Ojanen, Harri J.; Fussen, Didier; Pieroux, Didier; Demoulin, Philippe; Dekemper, Emmanuel; Vanhellemont, Filip

    2015-10-01

    PICASSO - A PICo-satellite for Atmospheric and Space Science Observations is an ESA project led by the Belgian Institute for Space Aeronomy, in collaboration with VTT, Clyde Space Ltd. (UK), and the Centre Spatial de Liège (BE). VTT Technical Research Centre of Finland Ltd. will deliver the Visible Spectral Imager for Occultation and Nightglow (VISION) for the PICASSO mission. The VISION targets primarily the observation of the Earth's atmospheric limb during orbital Sun occultation. By assessing the radiation absorption in the Chappuis band for different tangent altitudes, the vertical profile of the ozone is retrieved. A secondary objective is to measure the deformation of the solar disk so that stratospheric and mesospheric temperature profiles are retrieved by inversion of the refractive raytracing problem. Finally, occasional full spectral observations of polar auroras are also foreseen. The VISION design realized with commercial of the shelf (CoTS) parts is described. The VISION instrument is small, lightweight (~500 g), Piezo-actuated Fabry-Perot Interferometer (PFPI) tunable spectral imager operating in the visible and near-infrared (430 - 800 nm). The spectral resolution over the whole wavelength range will be better than 10 nm @ FWHM. VISION has is 2.5° x 2.5° total field of view and it delivers maximum 2048 x 2048 pixel spectral images. The sun image size is around 0.5° i.e. ~500 pixels. To enable fast spectral data image acquisition VISION can be operated with programmable image sizes. VTT has previously developed PFPI tunable filter based AaSI Spectral Imager for the Aalto-1 Finnish CubeSat. In VISION the requirements of the spectral resolution and stability are tighter than in AaSI. Therefore the optimization of the of the PFPI gap control loop for the operating temperature range and vacuum conditions has to be improved. VISION optical, mechanical and electrical design is described.

  18. Can spectral-spatial image segmentation be used to discriminate experimental burn wounds?

    NASA Astrophysics Data System (ADS)

    Paluchowski, Lukasz A.; Nordgaard, Håvard B.; Bjorgan, Asgeir; Hov, Håkon; Berget, Sissel M.; Randeberg, Lise L.

    2016-10-01

    Hyperspectral imaging (HSI) is a noncontact and noninvasive optical modality emerging the field of medical research. The goal of this study was to determine the ability of HSI and image segmentation to discriminate burn wounds in a preclinical porcine model. A heated brass rod was used to introduce burn wounds of graded severity in a pig model and a sequence of hyperspectral data was recorded up to 8-h postinjury. The hyperspectral images were processed by an unsupervised spectral-spatial segmentation algorithm. Segmentation was validated using results from histology. The proposed algorithm was compared to K-means segmentation and was found superior. The obtained segmentation maps revealed separated zones within the burn sites, indicating a variation in burn severity. The suggested image-processing scheme allowed mapping dynamic changes of spectral properties within the burn wounds over time. The results of this study indicate that unsupervised spectral-spatial segmentation applied on hyperspectral images can discriminate burn injuries of varying severity.

  19. Compact hybrid real-time hyperspectral imaging system with high effective spatial, spectral, and temporal resolution

    NASA Astrophysics Data System (ADS)

    Roth, Filip; Abbadi, Ahmad; Herman, Ondrej; Pavelek, Martin; Prenosil, Vaclav

    2016-10-01

    Medical endoscopes for image-guided surgery commonly use standard color image sensors, discarding any more detailed spectral information. Medical spectroscopy devices with various spectral working ranges are specialized to specific medical procedures and in general are not usable for image-guided surgery due to limitations in spatial or temporal resolution. In this paper, we present an initial demonstrator of hyperspectral endoscope, composed of two image sensors with complementing parameters. Using this hybrid approach, combining sensors with different spatial and spectral resolutions and spectral ranges, we obtain improved coverage of all the respective parameters. After digitally processing and merging the video streams, while maintaining the better features of both, we obtain an imaging system providing high effective spatial, spectral, and temporal resolution. The system is based on field programmable gate arrays. It provides real-time video output (60 Hz), which is usable for navigation during image-guided surgery. The flexible system architecture allows for an easy extension of the processing algorithms and enables minimal video signal latency. Physical dimensions and portability of the system are comparable to standard off-the-shelf medical endoscope cameras. The device can output both processed video and standard visible light video signals on one or more video outputs of the system. The resulting processed video signal obtained from the combined image sensor data greatly increases the amount of useful information available to the end user.

  20. Pigment Identification of Paintings Based on Kubelka-Munk Theory and Spectral Images

    NASA Astrophysics Data System (ADS)

    Moghareh Abed, Farhad

    The preservation of cultural heritage and treatment thereof are delicate responsibilities that demand the best possible technologies and extreme care to avoid any irreversible loss. It necessitates a deep understanding of constituent materials, along with the analytical methods and cutting-edge technologies. Considering this direction, the goal of this dissertation is to promote the conservation procedures by providing an applicable workflow for spectral-based pigment identification. The proposed pipeline is a novel and practical aid for museum conservators for many aspects, such as inpainting, treatment and archiving of artwork. Spectral-based pigment identification algorithms rely on accurate spectral data, a subtractive mixing model and an effective unmixing algorithm. In this dissertation, the spectral data were obtained using a spectral image acquisition system as a feasible and non-destructive technique. A liquid-crystal tunable filter (LCTF) and a CCD camera were used for spectral measurement of the painting. The spectral accuracy and precision of the LCTF-based spectral acquisition system were assessed and enhanced. Of the common factors affecting the acquisition performance, capturing geometry, LCTF angular dependencies and spectral characterization algorithm were new contributions to the traditional workflow. The complexity of subtractive mixtures limits the effective application of linear unmixing algorithms for pigment identification. Accordingly, a new linear modification of single-constant Kubelka-Munk theory was derived to enable the use of available linear spectral unmixing algorithms for paint mixtures. A selection of geometric and iterative-based unmixing algorithms was applied to the LCTF spectral images in the subtractive mixing space using the defined subtractive linear model. Final sets of primary pigments were improved employing a pre-existing database of common pigments as a tool for practical inpainting procedures. The pigment maps, showing

  1. Principal Components Analysis of Martian NIR Image Cubes to Retrieve Surface Spectral Endmembers

    NASA Astrophysics Data System (ADS)

    Klassen, David R.

    2016-07-01

    Presented here is a discussion of the complete principal components analysis (PCA) performed on all photometric NASA Infrared Telescope Facility (IRTF) NSFCAM spectral image sets from 1995-2001 and Mars Reconnaissance Orbiter (MRO) Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) spectral image sets from 2006-2008, detailing the similarities and differences and overall interpretation of the PC dimensional spaces. The purpose of the analysis is to use the PCA to recover surface spectral endmembers to be used in a full radiative transfer modeling program to recover ice cloud optical depths (and thus water content) over diurnal, seasonal, and interannual timescales. The PCA results show considerable consistency across all seasons, and can be optimized to increase the consistency through both spectral and geographic restrictions on the data.

  2. Methodology for diagnosing of skin cancer on images of dermatologic spots by spectral analysis

    PubMed Central

    Guerra-Rosas, Esperanza; Álvarez-Borrego, Josué

    2015-01-01

    In this paper a new methodology for the diagnosing of skin cancer on images of dermatologic spots using image processing is presented. Currently skin cancer is one of the most frequent diseases in humans. This methodology is based on Fourier spectral analysis by using filters such as the classic, inverse and k-law nonlinear. The sample images were obtained by a medical specialist and a new spe