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Sample records for confocal mosaicing microscopy

  1. Multimodal confocal mosaicing microscopy: an emphasis on squamous cell carcinoma

    NASA Astrophysics Data System (ADS)

    Chen, Nathaniel W.; Sensibaugh, Jordan; Ardeshiri, Ardaland; Blanchard, Adam; Jacques, Steven; Gareau, Daniel

    2010-02-01

    Our previous study reported a sensitivity of 96.6% and a specificity of 89.2% in rapidly detecting Basal Cell Carcinomas (BCCs) when nuclei were stained with acridine orange. Squamous Cell Carcinomas (SCCs) and infiltrative BCCs remain difficult to detect. More complete screening can be achieved utilizing both acridine orange for nuclei staining and eosin for cytoplasmic contrast, using two lasers to excite the two stains independently. Nuclear fluorescence is achieved by staining with acridine orange (0.5mM, 60 s), and cytoplasmic fluorescence is achieved by staining with eosin working solution (30 s). This work shows good morphological contrast of SCC and infiltrative BCC with eosin, acridine orange, and reflectance, and presents a means for rapid SCC and infiltrative BCC detection in fresh skin excisions using multimodal confocal microscopy. In addition, digital staining is shown to effectively simulate hematoxylin and eosin (H&E) histology with confocal mosaics.

  2. Strip mosaicing confocal microscopy for rapid imaging over large areas of excised tissue

    NASA Astrophysics Data System (ADS)

    Abeytunge, Sanjee; Li, Yongbiao; Larson, Bjorg; Peterson, Gary; Toledo-Crow, Ricardo; Rajadhyaksha, Milind

    2012-03-01

    Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in fresh tissue, without the processing that is required for conventional pathology. Previously, basal cell carcinoma margins were detected by mosaicing of confocal images of 12 x 12 mm2 of excised tissue from Mohs surgery. This mosaicing took 9 minutes. Recently we reported the initial feasibility of a faster approach called "strip mosaicing" on 10 x 10 mm2 of tissue that was demonstrated in 3 minutes. In this paper we report further advances in instrumentation and software. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Thus, strip mosaicing confocal microscopy may serve as an adjunct to pathology for imaging tumor margins to guide surgery.

  3. Sensitivity and Specificity for Detecting Basal Cell Carcinomas in Mohs Excisions with Confocal Fluorescence Mosaicing Microscopy

    PubMed Central

    Gareau, Daniel S.; Karen, Julie K.; Dusza, Stephen W.; Tudisco, Marie; Nehal, Kishwer S.; Rajadhyaksha, Milind

    2009-01-01

    Recent studies have demonstrated the ability of confocal fluorescence mosaicing microscopy to rapidly detect basal cell carcinomas (BCCs) directly in thick and fresh Mohs surgical excisions. Mosaics of confocal images display large areas of tissue with high resolution and magnification equivalent to 2X, which is the standard magnification when examining pathology. Comparison of mosaics to Mohs frozen histopathology was shown to be excellent for all types of BCCs. However, the comparisons in the previous studies were visual and qualitative. In this paper, we report the results of a semi-quantitative preclinical study in which forty-five confocal mosaics were blindly evaluated for the presence (or absence) of BCC tumor. The evaluations were by two clinicians: a senior Mohs surgeon, with prior expertise in interpreting confocal images, and a novice Mohs fellow, with limited experience. The blinded evaluation was compared to the gold standard of frozen histopathology. BCCs were detected with an overall sensitivity of 96.6%, specificity of 89.2%, positive predictive value of 93.0% and negative predictive value of 94.7%. The results demonstrate the potential clinical utility of confocal mosaicing microscopy toward rapid surgical pathology-at-the-bedside to expedite and guide surgery. PMID:19566305

  4. Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue

    NASA Astrophysics Data System (ADS)

    Abeytunge, Sanjee; Li, Yongbiao; Larson, Bjorg; Peterson, Gary; Seltzer, Emily; Toledo-Crow, Ricardo; Rajadhyaksha, Milind

    2013-06-01

    Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12 mm2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called "strip mosaicing," which was demonstrated on a 10-×-10 mm2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10 mm2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5 cm2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

  5. Confocal mosaicing microscopy in skin excisions: a demonstration of rapid surgical pathology

    PubMed Central

    Gareau, D.S.; Patel, Y.G.; Li, Y.; Aranda, I.; Halpern, A.C.; Nehal, K.S.; Rajadhyaksha, M.

    2009-01-01

    Summary Precise micro-surgical removal of tumour with minimal damage to the surrounding normal tissue requires a series of excisions, each guided by an examination of frozen histology of the previous. An example is Mohs surgery for the removal of basal cell carcinomas (BCCs) in skin. The preparation of frozen histology is labour-intensive and slow. Confocal microscopy may enable rapid detection of tumours directly in surgical excisions with minimal need for frozen histology. Mosaicing of images enables observation of nuclear and cellular morphology in large areas of surgically excised tissue. In skin, the use of 10–1% acetic acid as a reflectance contrast agent brightens nuclei in 0.5–5 min and enhances nuclear-to-dermis contrast and detectability of BCCs. A tissue fixture was engineered for precisely mounting surgical excisions to enable mosaicing of 36 × 36 images to create a field of view of 12 × 12 mm. This large field of view displays the excision at 2× magnification, similar to that routinely used by Mohs surgeons when examining frozen histology. Comparison of mosaics to histology demonstrates detectability of BCCs. Confocal mosaicing presently requires 9 min, instead of 20–45 min per excision for preparing frozen histology, and thus may provide a means for rapid pathology-at-the-bedside to expedite and guide surgery. PMID:19196421

  6. A machine learning method for identifying morphological patterns in reflectance confocal microscopy mosaics of melanocytic skin lesions in-vivo

    NASA Astrophysics Data System (ADS)

    Kose, Kivanc; Alessi-Fox, Christi; Gill, Melissa; Dy, Jennifer G.; Brooks, Dana H.; Rajadhyaksha, Milind

    2016-02-01

    We present a machine learning algorithm that can imitate the clinicians qualitative and visual process of analyzing reflectance confocal microscopy (RCM) mosaics at the dermal epidermal junction (DEJ) of skin. We divide the mosaics into localized areas of processing, and capture the textural appearance of each area using dense Speeded Up Robust Feature (SURF). Using these features, we train a support vector machine (SVM) classifier that can distinguish between meshwork, ring, clod, aspecific and background patterns in benign conditions and melanomas. Preliminary results on 20 RCM mosaics labeled by expert readers show classification with 55 - 81% sensitivity and 81 - 89% specificity in distinguishing these patterns.

  7. Implementation of fluorescence confocal mosaicing microscopy by "early adopter" Mohs surgeons: a review of recent progress

    NASA Astrophysics Data System (ADS)

    Jain, Manu; Rajadhyaksha, Milind; Nehal, Kishwer

    2016-03-01

    Confocal mosaicing microscopy (CMM) enables rapid imaging of large areas of fresh tissue ex vivo without the processing that is necessary for conventional histology. When performed with fluorescence mode using acridine orange (nuclear specific dye) it enhances nuclei-to-dermis contrast that enables detection of all types of BCCs including thin strands of infiltrative basal cell carcinomas (BCCs). Thus far, this technique has been mostly validated in research setting for the analysis of BCC tumor margins. Recently, CMM has been adopted and implemented in real clinical settings by some surgeons as an alternative tool to frozen section (FS) during Mohs surgery. In this review article we summarize the development of CMM guided imaging of ex vivo tissues from bench to bedside. We also present its current state of application in routine clinical workflow not only for the assessment of BCC margin but also for other skin cancers such as melanoma, SCC, and some infectious diseases where FS is not routinely performed. Lastly, we also discuss the potential limitations of this technology as well as future developments. As this technology advances further, it may serve as an adjunct to standard histology and enable rapid surgical pathology of skin cancers at the bedside.

  8. Fluorescence confocal mosaicing microscopy of basal cell carcinomas ex vivo: demonstration of rapid surgical pathology with high sensitivity and specificity

    NASA Astrophysics Data System (ADS)

    Gareau, Daniel S.; Karen, Julie K.; Dusza, Stephen W.; Tudisco, Marie; Nehal, Kishwer S.; Rajadhyaksha, Milind

    2009-02-01

    Mohs surgery, for the precise removal of basal cell carcinomas (BCCs), consists of a series of excisions guided by the surgeon's examination of the frozen histology of the previous excision. The histology reveals atypical nuclear morphology, identifying cancer. The preparation of frozen histology is accurate but labor-intensive and slow. Nuclear pathology can be achieved by staining with acridine orange (1 mM, 20 s) BCCs in Mohs surgical skin excisions within 5-9 minutes, compared to 20-45 for frozen histology. For clinical utility, images must have high contrast and high resolution. We report tumor contrast of 10-100 fold over the background dermis and submicron (diffraction limited) resolution over a cm field of view. BCCs were detected with an overall sensitivity of 96.6%, specificity of 89.2%, positive predictive value of 93.0% and negative predictive value of 94.7%. The technique was therefore accurate for normal tissue as well as tumor. We conclude that fluorescence confocal mosaicing serves as a sensitive and rapid pathological tool. Beyond Mohs surgery, this technology may be extended to suit other pathological needs with the development of new contrast agents. The technique reported here accurately detects all subtypes of BCC in skin excisions, including the large nodular, small micronodular, and tiny sclerodermaform tumors. However, this technique may be applicable to imaging tissue that is larger, more irregular and of various mechanical compliances with further engineering of the tissue mounting and staging mechanisms.

  9. Confocal microscopy and exfoliative cytology

    PubMed Central

    Reddy, Shyam Prasad; Ramani, Pratibha; Nainani, Purshotam

    2013-01-01

    Context: Early detection of potentially malignant lesions and invasive squamous-cell carcinoma in the oral cavity could be greatly improved through techniques that permit visualization of subtle cellular changes indicative of the neoplastic transformation process. One such technique is confocal microscopy. Combining rapidity with reliability, an innovative idea has been put forward using confocal microscope in exfoliative cytology. Aims: The main objective of this study was to assess confocal microscopy for cytological diagnosis and the results were compared with that of the standard PAP stain. Settings and Design: Confocal microscope, acridine orange (AO) stain, PAP (Papanicolaou) stain. The study was designed to assess confocal microscopy for cytological diagnosis. In the process, smears of patients with (clinically diagnosed and/or suspected) oral squamous cell carcinoma as well as those of controls (normal people) were stained with acridine orange and observed under confocal microscope. The results were compared with those of the standard PAP method. Materials and Methods: Samples of buccal mucosa smears from normal patients and squamous cell carcinoma patients were made, fixed in 100% alcohol, followed by AO staining. The corresponding set of smears was stained with PAP stain using rapid PAP stain kit. The results obtained were compared with those obtained with AO confocal microscopy. Results: The study had shown nuclear changes (malignant cells) in the smears of squamous cell carcinoma patients as increased intensity of fluorescence of the nucleus, when observed under confocal microscope. Acridine orange confocal microscopy showed good amount of sensitivity and specificity (93%) in identifying malignant cells in exfoliative cytological smears. Conclusion: Confocal microscopy was found to have good sensitivity in the identification of cancer (malignant) cells in exfoliative cytology, at par with the PAP method. The rapidity of processing and screening a

  10. Confocal microscopy in microgravity research

    NASA Astrophysics Data System (ADS)

    Goede, A. P. H.; Brakenhoff, G. J.; Woldringh, C. L.; Aalders, J. W. G.; Imhof, J. P.; van Kralingen, P.; Mels, W. A.; Schreinemakers, P.; Zegers, A.

    We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.

  11. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY

    EPA Science Inventory

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

  12. Confocal microscopy of skin cancers: Translational advances toward clinical utility

    PubMed Central

    Rajadhyaksha, Milind

    2014-01-01

    Recent advances in translational research in and technology for confocal microscopy of skin cancers, toward clinical applications, are described. Advances in translational research are in diagnosis of melanoma in vivo, pre-operative mapping of lentigo maligna melanoma margins to guide surgery and intra-operative imaging of residual basal cell carcinomas to guide shave-biopsy. Advances in technology include mosaicing microscopy for detection of basal cell carcinomas in large areas of excised tissue, toward rapid pathology-at-the-bedside, and development of small, simple and low-cost line-scanning confocal microscopes for worldwide use in diverse primary healthcare settings. Current limitations and future opportunities and challenges for both clinicians and technologists are discussed. PMID:19964286

  13. Multidepth imaging by chromatic dispersion confocal microscopy

    NASA Astrophysics Data System (ADS)

    Olsovsky, Cory A.; Shelton, Ryan L.; Saldua, Meagan A.; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

    2012-03-01

    Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.

  14. Multimodal confocal mosaics enable high sensitivity and specificity in screening of in situ squamous cell carcinoma

    NASA Astrophysics Data System (ADS)

    Grados Luyando, Maria del Carmen; Bar, Anna; Snavely, Nicholas; Jacques, Steven; Gareau, Daniel S.

    2014-02-01

    Screening cancer in excision margins with confocal microscopy may potentially save time and cost over the gold standard histopathology (H and E). However, diagnostic accuracy requires sufficient contrast and resolution to reveal pathological traits in a growing set of tumor types. Reflectance mode images structural details due to microscopic refractive index variation. Nuclear contrast with acridine orange fluorescence provides enhanced diagnostic value, but fails for in situ squamous cell carcinoma (SCC), where the cytoplasm is important to visualize. Combination of three modes [eosin (Eo) fluorescence, reflectance (R) and acridine orange (AO) fluorescence] enable imaging of cytoplasm, collagen and nuclei respectively. Toward rapid intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaics can image wide surgical margins (~1cm) with sub-cellular resolution and mimic the appearance of conventional H and E. Absorption contrast is achieved by alternating the excitation wavelength: 488nm (AO fluorescence) and 532nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H and E, enabling detection of the carcinoma in situ in the epidermal layer The sum mosaic Eo+R is false-colored pink to mimic eosins' appearance in H and E, while the AO mosaic is false-colored purple to mimic hematoxylins' appearance in H and E. In this study, mosaics of 10 Mohs surgical excisions containing SCC in situ and 5 containing only normal tissue were subdivided for digital presentation equivalent to 4X histology. Of the total 16 SCC in situ multimodal mosaics and 16 normal cases presented, two reviewers made 1 and 2 (respectively) type-2 errors (false positives) but otherwise scored perfectly when using the confocal images to screen for the presence of SCC in situ as compared to the gold standard histopathology. Limitations to precisely mimic H and E included occasional elastin staining by AO. These results suggest that

  15. Confocal multiview light-sheet microscopy

    PubMed Central

    Medeiros, Gustavo de; Norlin, Nils; Gunther, Stefan; Albert, Marvin; Panavaite, Laura; Fiuza, Ulla-Maj; Peri, Francesca; Hiiragi, Takashi; Krzic, Uros; Hufnagel, Lars

    2015-01-01

    Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage. PMID:26602977

  16. Confocal microscopy imaging of solid tissue

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ...

  17. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: AXIAL RESOLUTION

    EPA Science Inventory

    Abstract

    Confocal Microscopy System Performance: Axial resolution.
    Robert M. Zucker, PhD

    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Re...

  18. Confocal filtering in cathodoluminescence microscopy of nanostructures

    SciTech Connect

    Narváez, Angela C. E-mail: j.p.hoogenboom@tudelft.nl; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P. E-mail: j.p.hoogenboom@tudelft.nl; Kruit, Pieter

    2014-06-23

    Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effective for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.

  19. Confocal Raman Microscopy in Pharmaceutical Development

    NASA Astrophysics Data System (ADS)

    Haefele, Thomas F.; Paulus, Kurt

    There is a wide range of applications of confocal Raman microscopy in pharmaceutical development. It is a powerful tool to probe the distribution of components within a formulation, to characterize homogeneity of pharmaceutical samples, to determine solid state of drug substances and excipients and to characterize contaminations and foreign particulates. The information obtained by confocal Raman microscopy is extremely useful, sometimes even crucial, for drug substance design, for the development of solid and liquid formulations, as a tool for process analytics and for patent infringements and counterfeit analysis. In this chapter, those aspects and applications will be presented, focusing on solid drug formulations. This chapter will also reveal the advantages and demonstrate the synergies of Raman mapping as compared to similar imaging methods such as SEM/EDX, NIR and MIR imaging.

  20. Comprehensive volumetric confocal microscopy with adaptive focusing

    PubMed Central

    Kang, DongKyun; Yoo, Hongki; Jillella, Priyanka; Bouma, Brett E.; Tearney, Guillermo J.

    2011-01-01

    Comprehensive microscopy of distal esophagus could greatly improve the screening and surveillance of esophageal diseases such as Barrett’s esophagus by providing histomorphologic information over the entire region at risk. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that can be configured to image the entire distal esophagus by helically scanning the beam using optics within a balloon-centering probe. It is challenging to image the human esophagus in vivo with balloon-based SECM, however, because patient motion and anatomic tissue surface irregularities decenter the optics, making it difficult to keep the focus at a predetermined location within the tissue as the beam is scanned. In this paper, we present a SECM probe equipped with an adaptive focusing mechanism that can compensate for tissue surface irregularity and dynamic focal variation. A tilted arrangement of the objective lens is employed in the SECM probe to provide feedback signals to an adaptive focusing mechanism. The tilted configuration also allows the probe to obtain reflectance confocal data from multiple depth levels, enabling the acquisition of three-dimensional volumetric data during a single scan of the probe. A tissue phantom with a surface area of 12.6 cm2 was imaged using the new SECM probe, and 8 large-area reflectance confocal microscopy images were acquired over the depth range of 56 μm in 20 minutes. Large-area SECM images of excised swine small intestine tissue were also acquired, enabling the visualization of villous architecture, epithelium, and lamina propria. The adaptive focusing mechanism was demonstrated to enable acquisition of in-focus images even when the probe was not centered and the tissue surface was irregular. PMID:21698005

  1. Optimal pupil design for confocal microscopy

    NASA Astrophysics Data System (ADS)

    Patel, Yogesh G.; Rajadhyaksha, Milind; DiMarzio, Charles A.

    2010-02-01

    Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current instruments are large, complex, and expensive. A simpler, confocal line-scanning microscope may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A confocal reflectance microscope may use a beamsplitter, transmitting and detecting through the pupil, or a divided pupil, or theta configuration, with half used for transmission and half for detection. The divided pupil may offer better sectioning and contrast. We present a Fourier optics model and compare the on-axis irradiance of a confocal point-scanning microscope in both pupil configurations, optimizing the profile of a Gaussian beam in a circular or semicircular aperture. We repeat both calculations with a cylindrical lens which focuses the source to a line. The variable parameter is the fillfactor, h, the ratio of the 1/e2 diameter of the Gaussian beam to the diameter of the full aperture. The optimal values of h, for point scanning are 0.90 (full) and 0.66 for the half-aperture. For line-scanning, the fill-factors are 1.02 (full) and 0.52 (half). Additional parameters to consider are the optimal location of the point-source beam in the divided-pupil configuration, the optimal line width for the line-source, and the width of the aperture in the divided-pupil configuration. Additional figures of merit are field-of-view and sectioning. Use of optimal designs is critical in comparing the experimental performance of the different configurations.

  2. Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

    PubMed Central

    Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

    2008-01-01

    Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

  3. Combined Confocal and Magnetic Resonance Microscopy

    SciTech Connect

    Wind, Robert A.; Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Daly, Don S.; Holtom, Gary R.; Thrall, Brian D.; Weber, Thomas J.

    2002-05-12

    Confocal and magnetic resonance microscopy are both used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each of the microscopes individually. In this paper two configurations of a combined confocal and magnetic resonance microscope described. In both cases the sample compartment is part of a temperature regulated perfusion system. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas with the second configuration monolayers of mammalian cells can be investigated . Combined images are shown of Xenopus laevis frog oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.

  4. Digital confocal microscopy through a multimode fiber.

    PubMed

    Loterie, Damien; Farahi, Salma; Papadopoulos, Ioannis; Goy, Alexandre; Psaltis, Demetri; Moser, Christophe

    2015-09-01

    Acquiring high-contrast optical images deep inside biological tissues is still a challenging problem. Confocal microscopy is an important tool for biomedical imaging since it improves image quality by rejecting background signals. However, it suffers from low sensitivity in deep tissues due to light scattering. Recently, multimode fibers have provided a new paradigm for minimally invasive endoscopic imaging by controlling light propagation through them. Here we introduce a combined imaging technique where confocal images are acquired through a multimode fiber. We achieve this by digitally engineering the excitation wavefront and then applying a virtual digital pinhole on the collected signal. In this way, we are able to acquire images through the fiber with significantly increased contrast. With a fiber of numerical aperture 0.22, we achieve a lateral resolution of 1.5µm, and an axial resolution of 12.7µm. The point-scanning rate is currently limited by our spatial light modulator (20Hz). PMID:26368478

  5. Building large mosaics of confocal edomicroscopic images using visual servoing.

    PubMed

    Rosa, Benoît; Erden, Mustafa Suphi; Vercauteren, Tom; Herman, Benoît; Szewczyk, Jérôme; Morel, Guillaume

    2013-04-01

    Probe-based confocal laser endomicroscopy provides real-time microscopic images of tissues contacted by a small probe that can be inserted in vivo through a minimally invasive access. Mosaicking consists in sweeping the probe in contact with a tissue to be imaged while collecting the video stream, and process the images to assemble them in a large mosaic. While most of the literature in this field has focused on image processing, little attention has been paid so far to the way the probe motion can be controlled. This is a crucial issue since the precision of the probe trajectory control drastically influences the quality of the final mosaic. Robotically controlled motion has the potential of providing enough precision to perform mosaicking. In this paper, we emphasize the difficulties of implementing such an approach. First, probe-tissue contacts generate deformations that prevent from properly controlling the image trajectory. Second, in the context of minimally invasive procedures targeted by our research, robotic devices are likely to exhibit limited quality of the distal probe motion control at the microscopic scale. To cope with these problems visual servoing from real-time endomicroscopic images is proposed in this paper. It is implemented on two different devices (a high-accuracy industrial robot and a prototype minimally invasive device). Experiments on different kinds of environments (printed paper and ex vivo tissues) show that the quality of the visually servoed probe motion is sufficient to build mosaics with minimal distortion in spite of disturbances. PMID:23192481

  6. EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: PRETTY PICTURES OR CONFOCAL QA

    EPA Science Inventory

    Evaluation of confocal microscopy system performance: Pretty pictures or confocal QA?

    Robert M. Zucker

    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, N...

  7. Digital differential confocal microscopy based on spatial shift transformation.

    PubMed

    Liu, J; Wang, Y; Liu, C; Wilson, T; Wang, H; Tan, J

    2014-11-01

    Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen. PMID:25303106

  8. Confocal microscopy of the living eye.

    PubMed

    Cavanagh, H D; Jester, J V; Essepian, J; Shields, W; Lemp, M A

    1990-01-01

    Confocal microscopy is an imaging paradigm that allows optical sectioning of almost any material with increased axial and lateral spatial resolution and better image contrast. We have applied this technology to the study of the living eye of cats, albino rabbits, and humans. The technique allows in vivo, noninvasive, real time images of the eye at magnifications (630x) which allow resolution of anatomical detail at the cellular level. In this paper we report details of our current instrument techniques and some of our results. The past development, present state-of-the-art, and projected future advances and applications of this novel microscopy are discussed. Preliminary observations are reported for all layers of the cornea, the limbus, and wound-healing responses in single animals. PMID:2407380

  9. Characterization of Polymer Blends: Optical Microscopy (*Polarized, Interference and Phase Contrast Microscopy*) and Confocal Microscopy

    SciTech Connect

    Ramanathan, Nathan Muruganathan; Darling, Seth B.

    2015-01-01

    Chapter 15 surveys the characterization of macro, micro and meso morphologies of polymer blends by optical microscopy. Confocal Microscopy offers the ability to view the three dimensional morphology of polymer blends, popular in characterization of biological systems. Confocal microscopy uses point illumination and a spatial pinhole to eliminate out-of focus light in samples that are thicker than the focal plane.

  10. Automated cellular pathology in noninvasive confocal microscopy

    NASA Astrophysics Data System (ADS)

    Ting, Monica; Krueger, James; Gareau, Daniel

    2014-03-01

    A computer algorithm was developed to automatically identify and count melanocytes and keratinocytes in 3D reflectance confocal microscopy (RCM) images of the skin. Computerized pathology increases our understanding and enables prevention of superficial spreading melanoma (SSM). Machine learning involved looking at the images to measure the size of cells through a 2-D Fourier transform and developing an appropriate mask with the erf() function to model the cells. Implementation involved processing the images to identify cells whose image segments provided the least difference when subtracted from the mask. With further simplification of the algorithm, the program may be directly implemented on the RCM images to indicate the presence of keratinocytes in seconds and to quantify the keratinocytes size in the en face plane as a function of depth. Using this system, the algorithm can identify any irregularities in maturation and differentiation of keratinocytes, thereby signaling the possible presence of cancer.

  11. Corneal In Vivo Confocal Microscopy: Clinical Applications.

    PubMed

    You, Jae Young; Botelho, Paul J

    2016-01-01

    In vivo confocal microscopy (IVCM) has become a widely accepted imaging technique to study the human living cornea. It provides a unique opportunity to visualize the corneal tissue at the cellular level without damage and longitudinally observe its pathologic and normative changes. With rapidly evolving technology, there has been an abundance of interest in maximizing its potential to better understand the human cornea in health and disease. This is evidenced by a growing literature analyzing acquired and inherited corneal and also systemic diseases using corneal IVCM. This article provides a narrative review of IVCM and its applications. [Full article available at http://rimed.org/rimedicaljournal-2016-06.asp, free with no login]. PMID:27247970

  12. Evaluation of reflectance confocal microscopy in dermatophytosis.

    PubMed

    Hui, Dai; Xue-cheng, Sun; Ai-e, Xu

    2013-03-01

    Traditional diagnostic testing for dermatophyte infection currently requires skin scraping for light microscopy and/or fungal culture or skin biopsy. Immunofluorescent microscopy can also be used with calcofluor stain. All of these tests can be time-consuming to perform, require a waiting period for results and are invasive. This study aimed to define the in vivo reflectance confocal microscopy (RCM) features of superficial cutaneous fungal infections and to analyse concordance with microscopic examination. Totally, 45 patients, who were diagnosed with superficial cutaneous fungal infections according to the positive result of microscopic examination, were enrolled in this study. We selected three typical lesions examined by RCM, and then recorded the results. In the patients with the tinea manus and pedis, mycelium in stratum corneum was found by the RCM in 14 of 22 patients (14/22; 63.64%). In the patients with the tinea cruris, mycelium in stratum corneum was found by the RCM in 19 of 23 patients (19/23; 82.61%). RCM seems to be useful for microscopic evaluation of mycelium features and may have a scientific value in study of superficial cutaneous fungal infections. PMID:22963376

  13. Deep stroma investigation by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rossi, Francesca; Tatini, Francesca; Pini, Roberto; Valente, Paola; Ardia, Roberta; Buzzonetti, Luca; Canovetti, Annalisa; Malandrini, Alex; Lenzetti, Ivo; Menabuoni, Luca

    2015-03-01

    Laser assisted keratoplasty is nowadays largely used to perform minimally invasive surgery and partial thickness keratoplasty [1-3]. The use of the femtosecond laser enables to perform a customized surgery, solving the specific problem of the single patient, designing new graft profiles and partial thickness keratoplasty (PTK). The common characteristics of the PTKs and that make them eligible respect to the standard penetrating keratoplasty, are: the preservation of eyeball integrity, a reduced risk of graft rejection, a controlled postoperative astigmatism. On the other hand, the optimal surgical results after these PTKs are related to a correct comprehension of the deep stroma layers morphology, which can help in the identification of the correct cleavage plane during surgeries. In the last years some studies were published, giving new insights about the posterior stroma morphology in adult subjects [4,5]. In this work we present a study performed on two groups of tissues: one group is from 20 adult subjects aged 59 +/- 18 y.o., and the other group is from 15 young subjects, aged 12+/-5 y.o.. The samples were from tissues not suitable for transplant in patients. Confocal microscopy and Environmental Scanning Electron Microscopy (ESEM) were used for the analysis of the deep stroma. The preliminary results of this analysis show the main differences in between young and adult tissues, enabling to improve the knowledge of the morphology and of the biomechanical properties of human cornea, in order to improve the surgical results in partial thickness keratoplasty.

  14. Biological applications of confocal fluorescence polarization microscopy

    NASA Astrophysics Data System (ADS)

    Bigelow, Chad E.

    Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine

  15. Reflectance confocal microscopy for mucosal diseases.

    PubMed

    Cinotti, E; Labeille, B; Cambazard, F; Thuret, G; Gain, P; Perrot, J L

    2015-10-01

    Non-invasive, real-time microscopic imaging using in vivo reflectance confocal microscopy (RCM) has been demonstrated to be a useful tool for the evaluation of skin diseases and in particular for skin neoplasms. Recently, the RCM devices dedicated to the skin have also been applied to perform "virtual biopsies" of the oral, genital and ocular mucosa. In fact, mucosa is a sensitive area where non invasive imaging techniques are of high interest in order to spare biopsies and excisions. Mucosa is particularly suitable for RCM because of its thin or absent cornified layer and its thin epithelium that allows a deeper penetration of the laser with the consequent possibility of exploring deeper tissue levels. Besides, being useful for the diagnosis, RCM may be helpful to identify the area to be biopsied in case of large or multifocal lesions and may be regarded as a complementary technique for non invasive assessment of treatment efficacy. The RCM features of healthy mucosa are described and a revision of the literature of the mucosal diseases that can be diagnosed by RCM has been performed. PMID:26099354

  16. EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE

    EPA Science Inventory

    BACKGROUND. The confocal laser scanning microscope (CLSM) has enormous potential in many biological fields. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by ea...

  17. Video-Mosaicing of Reflectance Confocal Images For Rapid Examination of Large Areas of Skin In Vivo

    PubMed Central

    Kose, Kivanc; Cordova, Miguel; Duffy, Megan; Flores, Eileen S.; Brooks, Dana H.; Rajadhyaksha, Milind

    2015-01-01

    Background With reflectance confocal microscopy (RCM) imaging, skin cancers can be diagnosed in vivo and margins detected to guide treatment. Since the field of view of an RCM image is much smaller than the typical size of lesions, mosaicing approaches have been developed to display larger areas of skin. However, the current paradigm for RCM mosaicing in vivo is limited both in speed and to pre-selected rectangular-shaped small areas. Another approach, called “video-mosaicing,” enables higher speeds and real-time operator-selected areas of any size and shape, and will be more useful for RCM examination of skin in vivo. Objectives To demonstrate the feasibility and clinical potential of video-mosaicing of RCM images to rapidly display large areas of skin in vivo. Methods Thirteen videos of benign lesions, melanocytic cancers and residual basal cell carcinoma margins were collected on volunteer subjects with a handheld RCM scanner. The images from each video were processed and stitched into mosaics to display the entire area that was imaged. Results Acquisition of RCM videos covering 5.0–16.0 mm2 was performed in 20–60 seconds. The video-mosaics were visually determined to be of high quality for resolution, contrast and seamless contiguity, and the appearance of cellular-level and morphologic detail. Conclusion Video-mosaicing confocal microscopy, with real-time operator-choice of the shape and size of the area to be imaged, will enable rapid examination of large areas of skin in vivo. This approach may further advance noninvasive detection of skin cancer and, eventually, facilitate wider adoption of RCM imaging in the clinic. PMID:24720744

  18. CONFOCAL LASER SCANNING MICROSCOPY OF RAT FOLLICLE DEVELOPMENT

    EPA Science Inventory

    This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pr...

  19. Confocal microscopy via multimode fibers: fluorescence bandwidth

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2016-03-01

    We recently described a method for confocal reflection imaging through fibers, as a way to increase contrast when imaging unstained biological specimens. Using a transmission matrix, focused spots can be created at the distal end of a fiber. The backscattered field coming back from the sample can be filtered using optical correlation to obtain spatial selectivity in the detection. In this proceedings article, we briefly review the working principle of this method, and we discuss how the scheme could be adapted to confocal fluorescence imaging. In particular, we show simulations of the achievable detection bandwidth when using step-index multimode fibers as imaging devices.

  20. In Vivo Confocal Microscopy in Chloroquine-Induced Keratopathy

    PubMed Central

    Paladini, Iacopo; Menchini, Ugo; Mencucci, Rita

    2013-01-01

    In vivo confocal microscopy is becoming a mandatory examination to study corneal abnormalities such as drug deposits in systemic disease. A female diagnosed with fibromyalgia on systemic chloroquine for 9 months presented for an ophthalmic examination. Confocal microscopy was performed using the Confoscan 4 (Nidek Co. Ltd., Gamagori, Japan) and multiple highly reflective deposits in the epithelial basal cells were found, that were consistent with choloquine. Deposits were also present in the wing cell layer. In the anterior stroma these deposits were rare. Atypically shaped and branched nerves were also present in the anterior stroma. Corneal deposits of chloroquine can be evaluated by confocal microscopy. Confocal microscopy provides information on corneal metabolism and physiology. Chloroquine keratopathy can affect the anterior stroma in addition to the epithelium. PMID:23580857

  1. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: LASER POWER MEASUREMENTS

    EPA Science Inventory

    Laser power abstract
    The reliability of the confocal laser-scanning microscope (CLSM) to obtain intensity measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. The laser power test appears to be one ...

  2. An alternative method of promoter assessment by confocal laser scanning microscopy.

    PubMed

    Sahoo, Dipak K; Ranjan, Rajiv; Kumar, Deepak; Kumar, Alok; Sahoo, Bhabani S; Raha, Sumita; Maiti, Indu B; Dey, Nrisingha

    2009-10-01

    A rapid and useful method of promoter activity analysis using techniques of confocal laser scanning microscopy (CLSM) is described in the present study. The activities of some pararetroviral promoters such as CaMV35S (Cauliflower mosaic virus), FMVSgt3 (Figwort mosaic virus sub-genomic transcript) and MMVFLt12 (Mirabilis mosaic virus full-length transcript) coupled to GFP (green fluorescent protein) and GUS (beta-glucuronidase) reporter genes were determined simultaneously by the CLSM technique and other available conventional methods for reporter gene assay based on relevant biochemical and molecular approaches. Consistent and comparable results obtained by CLSM as well as by other conventional assay methods confirm the effectiveness of the CLSM approach for assessment of promoter activity. Hence the CLSM method can be suggested as an alternative way for promoter analysis on the basis of high throughput. PMID:19540268

  3. Fluorescence performance standards for confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rüttinger, Steffen; Kapusta, Peter; Völlkopf, Volker; Koberling, Felix; Erdmann, Rainer; Macdonald, Rainer

    2010-02-01

    State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics. The main quantities such performance standard must address are: • Spatial resolution • Sensitivity • Fluorescence lifetime To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the corresponding standards have to be ready made, easy to handle and to store. The measurement procedures necessary should be available on as many different setups as possible and the procedures involved in their deployment should be as easy as possible. To this end, we developed two performance standards to accomplish the mentioned goals: • Resolution reference • Combined molecular brightness and fluorescence lifetime reference The first one is based on sub-resolution sized Tetra-SpeckTM fluorescent beads or alternatively on single molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to ascertain durability and road capabilities.

  4. Detection limits of confocal surface plasmon microscopy

    PubMed Central

    Pechprasarn, Suejit; Somekh, Michael G.

    2014-01-01

    This paper applies rigorous diffraction theory to evaluate the minimum mass sensitivity of a confocal optical microscope designed to excite and detect surface plasmons operating on a planar metallic substrate. The diffraction model is compared with an intuitive ray picture which gives remarkably similar predictions. The combination of focusing the surface plasmons and accurate phase measurement mean that under favorable but achievable conditions detection of small numbers of molecules is possible, however, we argue that reliable detection of single molecules will benefit from the use of structured surfaces. System configurations needed to optimize performance are discussed. PMID:24940537

  5. Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

    NASA Astrophysics Data System (ADS)

    Gareau, Dan; Bar, Anna; Snaveley, Nicholas; Lee, Ken; Chen, Nathaniel; Swanson, Neil; Simpson, Eric; Jacques, Steve

    2012-06-01

    For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

  6. Tri-modal confocal mosaics detect residual invasive squamous cell carcinoma in Mohs surgical excisions

    PubMed Central

    Bar, Anna; Snaveley, Nicholas; Lee, Ken; Chen, Nathaniel; Swanson, Neil; Simpson, Eric; Jacques, Steve

    2012-01-01

    Abstract. For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues. PMID:22734774

  7. Confocal fluorescence microscopy for detection of cervical preneoplastic lesions

    NASA Astrophysics Data System (ADS)

    Sheikhzadeh, Fahime; Ward, Rabab K.; Carraro, Anita; Chen, Zhaoyang; van Niekerk, Dirk; MacAulay, Calum; Follen, Michele; Lane, Pierre; Guillaud, Martial

    2015-03-01

    We examined and established the potential of ex-vivo confocal fluorescence microscopy for differentiating between normal cervical tissue, low grade Cervical Intraepithelial Neoplasia (CIN1), and high grade CIN (CIN2 and CIN3). Our objectives were to i) use Quantitative Tissue Phenotype (QTP) analysis to quantify nuclear and cellular morphology and tissue architecture in confocal microscopic images of fresh cervical biopsies and ii) determine the accuracy of high grade CIN detection via confocal microscopy. Cervical biopsy specimens of colposcopically normal and abnormal tissues obtained from 15 patients were evaluated by confocal fluorescence microscopy. Confocal images were analyzed and about 200 morphological and architectural features were calculated at the nuclear, cellular, and tissue level. For the purpose of this study, we used four features to delineate disease grade including nuclear size, cell density, estimated nuclear-cytoplasmic (ENC) ratio, and the average of three nearest Delaunay neighbors distance (3NDND). Our preliminary results showed ENC ratio and 3NDND correlated well with histopathological diagnosis. The Spearman correlation coefficient between each of these two features and the histopathological diagnosis was higher than the correlation coefficient between colposcopic appearance and histopathological diagnosis. Sensitivity and specificity of ENC ratio for detecting high grade CIN were both equal to 100%. QTP analysis of fluorescence confocal images shows the potential to discriminate high grade CIN from low grade CIN and normal tissues. This approach could be used to help clinicians identify HGSILs in clinical settings.

  8. Reflectance confocal microscopy in infectious diseases.

    PubMed

    Cinotti, E; Labeille, B; Cambazard, F; Perrot, J L

    2015-10-01

    In vivo reflectance confocal microscope (RCM) is a high-resolution non-invasive imaging technique that was initially focused on the diagnosis of skin cancers. A rising number of other indications have been later described for the diagnosis and management of inflammatory and infectious dermatological disorders. RCM can identify cutaneous parasites that are not visible to naked eye such as Sarcoptes scabiei and Demodex folliculorum and it allows to better identify the different body parts of bigger parasites such as ticks. Fungal filaments can also be identified as elongated bright structures in the cutaneous upper layers. RCM cannot observe virus directly. However, the cytopathic effect associated with some virus can be recognized. In addition of being helpful for the diagnosis and follow-up after treatment, thanks to its non-invasiveness, RCM allows pathophysiological studies. PMID:26129682

  9. Visualizing Cochlear Mechanics Using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Ulfendahl, M.; Boutet de Monvel, J.; Fridberger, A.

    2003-02-01

    The sound-evoked vibration pattern of the hearing organ is based on complex mechanical interactions between different cellular structures. To explore the structural changes occurring within the organ of Corti during basilar-membrane motion, stepwise alterations of the scala tympani pressure were applied in an in vitro preparation of the guinea-pig temporal bone. Confocal images were acquired at each pressure level. In this way, the motion of several structures could be simultaneously observed with high resolution in a nearly intact system. Images were analyzed using a novel wavelet-based optical-flow estimation algorithm. Under the present experimental conditions, the reticular lamina moved as a stiff plate with a center of rotation in the region of the inner hair cells. The outer hair cells appeared non-rigid and the basal, synaptic regions of these cells displayed significant radial motion indicative of cellular bending and internal shearing.

  10. Chromatic confocal microscopy using staircase diffractive surface.

    PubMed

    Rayer, Mathieu; Mansfield, Daniel

    2014-08-10

    A chromatic confocal microscope (CCM) is a high-dynamic-range noncontact distance measurement sensor; it is based on a hyperchromatic lens. The vast majority of commercial CCMs use refractive-based chromatic dispersion to chromatically code the optical axis. This approach significantly limits the range of applications and performance of the CCM. In order to be a suitable alternative to a laser triangulation gauge and laser encoder, the performance of the CCM must be improved. In this paper, it is shown how hybrid aspheric diffractive (HAD) lenses can bring the CCM to its full potential by increasing the dynamic range by a factor of 2 and the resolution by a factor of 5 while passively athermizing and increasing the light throughput efficiency of the optical head [M. Rayer, U.S. patent 1122052.2 (2011)]. The only commercially suitable manufacturing process is single-point diamond turning. However, the optical power carried by the diffractive side of a hybrid aspheric diffractive lens is limited by the manufacturing process. A theoretical study of manufacturing losses has revealed that the HAD configuration with the highest diffraction efficiency is for a staircase diffractive surface (SDS). SDS lenses have the potential to reduce light losses associated with manufacturing limits by a factor of 5 without increasing surface roughness, allowing scalar diffraction-limited optical design with a diffractive element. PMID:25320920

  11. Confocal Raman microscopy of protein adsorbed in chromatographic particles.

    PubMed

    Xiao, Yuewu; Stone, Thomas; Bell, David; Gillespie, Christopher; Portoles, Marta

    2012-09-01

    Confocal Raman microscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure conformation and distribution of protein adsorbed in wetted chromatographic particles. Monoclonal antibody was loaded into the Fractogel EMD SO(3) (M) cation exchanger at 2 mS/cm or 10 mS/cm. Amide I and III frequencies in the Raman spectrum of the adsorbed protein suggest that there are no detectable changes of the original β-sheet conformation in the chromatographic particles. Protein depth profile measurements indicate that, when the conductivity is increased from 2 mS/cm to 10 mS/cm, there is a change in mass transport mechanism for protein adsorption, from the shrinking-core model to the homogeneous-diffusion model. In this study, the use of confocal Raman microscopy to measure protein distribution in chromatographic particles fundamentally agrees with previous confocal laser scanning microscopic investigations, but confocal Raman spectroscopy enjoys additional advantages: use of unlabeled protein to eliminate fluorescent labeling, ability for characterization of protein secondary structure, and ability for spectral normalization to provide a nondestructive experimental approach to correct light attenuation effects caused by refractive index (RI) mismatching in semiopaque chromatographic particles. PMID:22803776

  12. Video-rate scanning confocal microscopy and microendoscopy.

    PubMed

    Nichols, Alexander J; Evans, Conor L

    2011-01-01

    Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets, monitor dynamics in living cells, and visualize the three dimensional evolution of entire organisms. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo and are currently being applied to disease imaging and diagnosis in clinical settings. Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples. Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not

  13. Video-rate Scanning Confocal Microscopy and Microendoscopy

    PubMed Central

    Nichols, Alexander J.; Evans, Conor L.

    2011-01-01

    Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9. Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples. Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will

  14. Colloidal structural evolution of asphaltene studied by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Hung, Jannett; Castillo, Jimmy A.; Reyes, A.

    2004-10-01

    In this work, a detail analysis of the flocculation kinetic of asphaltenes colloidal particles has been carried out usng confocal microscopy. The colloidal structural evolution of the asphaltene flocculated has had varies postulated; however, the aggregation process of asphaltene is still not fully understood. In a recent paper, using Confocal microscope (homemade), we reported high-resolution micrographic images of asphaltenes flocculated and the correlation between crude oil stability and flocculation process. This technique permitted visualizes directly the physical nature of asphaltene flocculated. In this work, a detail analysis of the flocculation kinetic of asphaltene colloidal particles has been carried out using confocal microscopy. The physical nature of asphaltene flocculated from different crude oils is showed through of high-resolution image micrographies and its colloidal structural evolution.

  15. FOOD SURFACE TEXTURE MEASUREMENT USING REFLECTIVE CONFOCAL LASER SCANNING MICROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Confocal laser scanning microscopy (CLSM) was used in the reflection mode to characterize the surface texture (roughness) of sliced food surfaces. Sandpapers of grit size between 150 and 600 were used as the height reference to standardize the CLSM hardware settings. Sandpaper particle sizes were v...

  16. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

    The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

  17. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: QA TESTS, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

    Confocal Microscopy System Performance: QA tests, Quantitation and Spectroscopy.

    Robert M. Zucker 1 and Jeremy M. Lerner 2,
    1Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research Development, U.S. Environmen...

  18. Three-Dimensional Visualization of Interfacial Phenomena Using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Shieh, Ian C.

    Surfactants play an integral role in numerous functions ranging from stabilizing the emulsion in a favorite salad dressing to organizing the cellular components that make life possible. We are interested in lung surfactant, which is a mixture of lipids and proteins essential for normal respiration because it modulates the surface tension of the air-liquid interface of the thin fluid lining in the lungs. Through this surface tension modulation, lung surfactant ensures effortless lung expansion and prevents lung collapse during exhalation, thereby effecting proper oxygenation of the bloodstream. The function of lung surfactant, as well as numerous interfacial lipid systems, is not solely dictated by the behavior of materials confined to the two-dimensional interface. Rather, the distributions of materials in the liquid subphase also greatly influence the performance of interfacial films of lung surfactant. Therefore, to better understand the behavior of lung surfactant and other interfacial lipid systems, we require a three-dimensional characterization technique. In this dissertation, we have developed a novel confocal microscopy methodology for investigating the interfacial phenomena of surfactants at the air-liquid interface of a Langmuir trough. Confocal microscopy provides the excellent combination of in situ, fast, three-dimensional visualization of multiple components of the lung surfactant system that other characterization techniques lack. We detail the solutions to the numerous challenges encountered when imaging a dynamic air-liquid interface with a high-resolution technique like confocal microscopy. We then use confocal microscopy to elucidate the distinct mechanisms by which a polyelectrolyte (chitosan) and nonadsorbing polymer (polyethylene glycol) restore the function of lung surfactant under inhibitory conditions mimicking the effects of lung trauma. Beyond this physiological model, we also investigate several one- and two-component interfacial films

  19. Confocal microscopy and variable-focal length microlenses

    NASA Astrophysics Data System (ADS)

    Mac Raighne, Aaron M.; Yang, Lisong; Dunbar, L. Andrea; McCabe, Eithne M.; Scharf, Toralf

    2004-07-01

    Confocal microscopy has a unique optical sectioning property which allows three-dimensional images at different depths. Use of a microlens array is a potential alternative to the Nipkow disk for parallel imaging with high throughput in real-time confocal microscopy. The use of variable-focal-length microlenses can provide a way to axially scan the foci electronically avoiding the inflexible mechanical movement of the lens or the sample. Here we demonstrate a combination of a variable-focal-length microlens array and a fiber optic bundle as a way to create a high throughput aperture array that would be potentially applied as confocal imaging in vivo biological specimens. Variable focal length microlenses that we use consist of a liquid crystal film sandwiched between a pair of conductive substrates with patterned electrodes. The incident side of the microlens array was determined by examining the focus distribution in the axial direction. The variation of the focal length obtained by changing the voltage and corresponding focus intensity were measured through a conventional microscope. Meanwhile, the fiber bundle was characterized by coupling with either coherent or incoherent light source. We use the fiber bundle as both a multiple aperture and an image-carrying element and combine it with a microlens array to built up a confocal system. Axial responses are measured in two optical arrangements as a route to investigate endoscope potential.

  20. Rapid confocal imaging of large areas of excised tissue with strip mosaicing

    PubMed Central

    Abeytunge, Sanjee; Li, Yongbiao; Larson, Bjorg; Toledo-Crow, Ricardo; Rajadhyaksha, Milind

    2011-01-01

    Imaging large areas of tissue rapidly and with high resolution may enable rapid pathology at the bedside. The limited field of view of high-resolution microscopes requires the merging of multiple images that are taken sequentially to cover a large area. This merging or mosaicing of images requires long acquisition and processing times, and produces artifacts. To reduce both time and artifacts, we developed a mosaicing method on a confocal microscope that images morphology in large areas of excised tissue with sub-cellular detail. By acquiring image strips with aspect ratios of 10:1 and higher (instead of the standard ∼1:1) and “stitching” them in software, our method images 10×10 mm2 area of tissue in about 3 min. This method, which we call “strip mosaicing,” is currently three times as fast as our previous method. PMID:21639560

  1. Confocal device and application strategies for endoluminal optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    George, Markus; Schnieder, Ludger; Buess, Gerhard F.

    2003-10-01

    While endoscopic optical coherence tomography has been established successfully in vivo ,implementation of endoluminal optical coherence microscopy remains demanding,s suitable confocal probe is lacking. A miniaturized confocal laser scanning microscope is presented,which fulfills the requirements for endoluminal optical coherence microscopy. First,imaging experience gained for optical coherence microscopy of nimal gastrointestinal tissue samples is described. For this purpose,laboratory scale optical coherence microscope with an image acquisition time of 1min 30 s was employed. Cellular membranes can be identified throughout the gastrointestinal organs. Frequency domain image analysis can be used to distinguish columnar from squamous epithelium. Profilometric information on sample surfaces can be obtained directly as isophase lines. Second, the miniaturized confocal laser scanning microscope is characterized. Having an effective diameter of 25 mm, it houses single-mode optical fiber,scanning mirror and an objective lens. The micro-electro-mechanical mirror with gimballed suspension allows two dimensional scanning without introducing an optical path difference. The sinusoidal movement of both axes has to be considered to approximate cartesian image coordinates. Field geometry is illustrated s function of excitation amplitude and frequency. Acceptable image quality is chieved for frame rate of 0.5 Hz. A strategy to position the focal plane axially within the sample volume is discussed.

  2. Study of liquid jet instability by confocal microscopy.

    PubMed

    Yang, Lisong; Adamson, Leanne J; Bain, Colin D

    2012-07-01

    The instability of a liquid microjet was used to measure the dynamic surface tension of liquids at the surface ages of ≤1 ms using confocal microscopy. The reflected light from a laser beam at normal incidence to the jet surface is linear in the displacement of the surface near the confocal position, leading to a radial resolution of 4 nm and a dynamic range of 4 μm in the surface position, thus permitting the measurement of amplitude of oscillation at the very early stage of jet instability. For larger oscillations outside the linear region of the confocal response, the swell and neck position of the jet can be located separately and the amplitude of oscillation determined with an accuracy of 0.2 μm. The growth rate of periodically perturbed water and ethanol∕water mixture jets with a 100-μm diameter nozzle and mean velocity of 5.7 m s(-1) has been measured. The dynamic surface tension was determined from the growth rate of the instability with a linear, axisymmetric, constant property model. Synchronisation of the confocal imaging system with the perturbation applied to the jet permitted a detailed study of the temporal evolution of the neck into a ligament and eventually into a satellite drop. PMID:22852668

  3. Study of liquid jet instability by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Lisong; Adamson, Leanne J.; Bain, Colin D.

    2012-07-01

    The instability of a liquid microjet was used to measure the dynamic surface tension of liquids at the surface ages of ≤1 ms using confocal microscopy. The reflected light from a laser beam at normal incidence to the jet surface is linear in the displacement of the surface near the confocal position, leading to a radial resolution of 4 nm and a dynamic range of 4 μm in the surface position, thus permitting the measurement of amplitude of oscillation at the very early stage of jet instability. For larger oscillations outside the linear region of the confocal response, the swell and neck position of the jet can be located separately and the amplitude of oscillation determined with an accuracy of 0.2 μm. The growth rate of periodically perturbed water and ethanol/water mixture jets with a 100-μm diameter nozzle and mean velocity of 5.7 m s-1 has been measured. The dynamic surface tension was determined from the growth rate of the instability with a linear, axisymmetric, constant property model. Synchronisation of the confocal imaging system with the perturbation applied to the jet permitted a detailed study of the temporal evolution of the neck into a ligament and eventually into a satellite drop.

  4. Confocal microscopy patterns in nonmelanoma skin cancer and clinical applications.

    PubMed

    González, S; Sánchez, V; González-Rodríguez, A; Parrado, C; Ullrich, M

    2014-06-01

    Reflectance confocal microscopy is currently the most promising noninvasive diagnostic tool for studying cutaneous structures between the stratum corneum and the superficial reticular dermis. This tool gives real-time images parallel to the skin surface; the microscopic resolution is similar to that of conventional histology. Numerous studies have identified the main confocal features of various inflammatory skin diseases and tumors, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings. Confocal patterns and diagnostic algorithms have been shown to have high sensitivity and specificity in melanoma and nonmelanoma skin cancer. Possible present and future applications of this noninvasive technology are wide ranging and reach beyond its use in noninvasive diagnosis. This tool can also be used, for example, to evaluate dynamic skin processes that occur after UV exposure or to assess tumor response to noninvasive treatments such as photodynamic therapy. We explain the characteristic confocal features found in the main nonmelanoma skin tumors and discuss possible applications for this novel diagnostic technique in routine dermatology practice. PMID:24002008

  5. Parallel detection experiment of fluorescence confocal microscopy using DMD.

    PubMed

    Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

    2016-05-01

    Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. PMID:26331288

  6. Polarization conversion in confocal microscopy with radially polarized illumination.

    PubMed

    Tang, Wai Teng; Yew, Elijah Y S; Sheppard, Colin J R

    2009-07-15

    The effects of using radially polarized illumination in a confocal microscope are discussed, and the introduction of a polarization mode converter into the detection optics of the microscope is proposed. We find that with such a configuration, bright-field imaging can be performed without losing the resolution advantage of radially polarized illumination. The detection efficiency can be increased by three times without having to increase the pinhole radius and sacrificing the confocality of the system. Furthermore, the merits of such a setup are also discussed in relation to surface plasmon microscopy and single-molecule orientation studies, where the doughnut point spread function can be engineered into a single-lobed point spread function. PMID:19823530

  7. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  8. Multimodal confocal hyperspectral imaging microscopy with wavelength sweeping source

    NASA Astrophysics Data System (ADS)

    Kim, Young-Duk; Do, Dukho; Yoo, Hongki; Gweon, DaeGab

    2015-02-01

    There exist microscopes that are able to obtain the chemical properties of a sample, because there are some cases in which it is difficult to find out causality of a phenomenon by using only the structural information of a sample. Obtaining the chemical properties of a sample is important in biomedical imaging, because most biological phenomena include changes in the chemical properties of the sample. Hyperspectral imaging (HSI) is one of the popular imaging methods for characterizing materials and biological samples by measuring the reflectance or emission spectrum of the sample. Because all materials have a unique reflectance spectrum, it is possible to analyze material properties and detect changes in the chemical properties of a sample by measuring the spectral changes with respect to the original spectrum. Because of its ability to measure the spectrum of a sample, HSI is widely used in materials identification applications such as aerial reconnaissance and is the subject of various studies in microscopy. Although there are many advantages to using the method, conventional HSI has some limitations because of its complex configuration and slow speed. In this research we propose a new type of multimodal confocal hyperspectral imaging microscopy with fast image acquisition and a simple configuration that is capable of both confocal and HSI microscopies.

  9. Imaging intracellular protein dynamics by spinning disk confocal microscopy

    PubMed Central

    Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

    2012-01-01

    The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

  10. Robust mosaicing with correction of motion distortions and tissue deformations for in vivo fibered microscopy.

    PubMed

    Vercauteren, Tom; Perchant, Aymeric; Malandain, Grégoire; Pennec, Xavier; Ayache, Nicholas

    2006-10-01

    Real-time in vivo and in situ imaging at the cellular level can be achieved with fibered confocal microscopy. As interesting as dynamic sequences may be, there is a need for the biologist or physician to get an efficient and complete representation of the entire imaged region. For this demand, the potential of this imaging modality is enhanced by using video mosaicing techniques. Classical mosaicing algorithms do not take into account the characteristics of fibered confocal microscopy, namely motion distortions, irregularly sampled frames and non-rigid deformations of the imaged tissue. Our approach is based on a hierarchical framework that is able to recover a globally consistent alignment of the input frames, to compensate for the motion distortions and to capture the non-rigid deformations. The proposed global alignment scheme is seen as an estimation problem on a Lie group. We model the relationship between the motion and the motion distortions to correct for these distortions. An efficient scattered data approximation scheme is proposed both for the construction of the mosaic and to adapt the demons registration algorithm to our irregularly sampled inputs. Controlled experiments have been conducted to evaluate the performance of our algorithm. Results on several sequences acquired in vivo on both human and mouse tissue also demonstrate the relevance of our approach. PMID:16887375

  11. Atherosclerotic plaque detection by confocal Brillouin and Raman microscopies

    NASA Astrophysics Data System (ADS)

    Meng, Zhaokai; Basagaoglu, Berkay; Yakovlev, Vladislav V.

    2015-02-01

    Atherosclerosis, the development of intraluminal plaque, is a fundamental pathology of cardiovascular system and remains the leading cause of morbidity and mortality worldwide. Biomechanical in nature, plaque rupture occurs when the mechanical properties of the plaque, related to the morphology and viscoelastic properties, are compromised, resulting in intraluminal thrombosis and reduction of coronary blood flow. In this report, we describe the first simultaneous application of confocal Brillouin and Raman microscopies to ex-vivo aortic wall samples. Such a non-invasive, high specific approach allows revealing a direct relationship between the biochemical and mechanical properties of atherosclerotic tissue.

  12. High-resolution confocal microscopy using synchrotron radiation.

    PubMed

    van der Oord, C J; Jones, G R; Shaw, D A; Munro, I H; Levine, Y K; Gerritsen, H C

    1996-06-01

    A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2 optics allows for confocal imaging over the wavelength range 200-700 nm. This includes UV light, which is particularly suitable for high-resolution imaging. The results of test measurements using 290-nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real-time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9-dihydroxy-6H-benzofurol[3,2-c][1]benzo-pyran-6-one (Chem. Abstr. Reg. No. 479-13-0). The trivial name will be used throughout this paper. PMID:8801359

  13. Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope

    PubMed Central

    Dubra, Alfredo; Sulai, Yusufu; Norris, Jennifer L.; Cooper, Robert F.; Dubis, Adam M.; Williams, David R.; Carroll, Joseph

    2011-01-01

    The rod photoreceptors are implicated in a number of devastating retinal diseases. However, routine imaging of these cells has remained elusive, even with the advent of adaptive optics imaging. Here, we present the first in vivo images of the contiguous rod photoreceptor mosaic in nine healthy human subjects. The images were collected with three different confocal adaptive optics scanning ophthalmoscopes at two different institutions, using 680 and 775 nm superluminescent diodes for illumination. Estimates of photoreceptor density and rod:cone ratios in the 5°–15° retinal eccentricity range are consistent with histological findings, confirming our ability to resolve the rod mosaic by averaging multiple registered images, without the need for additional image processing. In one subject, we were able to identify the emergence of the first rods at approximately 190 μm from the foveal center, in agreement with previous histological studies. The rod and cone photoreceptor mosaics appear in focus at different retinal depths, with the rod mosaic best focus (i.e., brightest and sharpest) being at least 10 μm shallower than the cones at retinal eccentricities larger than 8°. This study represents an important step in bringing high-resolution imaging to bear on the study of rod disorders. PMID:21750765

  14. Segmentation of skin strata in reflectance confocal microscopy depth stacks

    NASA Astrophysics Data System (ADS)

    Hames, Samuel C.; Ardigò, Marco; Soyer, H. Peter; Bradley, Andrew P.; Prow, Tarl W.

    2015-03-01

    Reflectance confocal microscopy is an emerging tool for imaging human skin, but currently requires expert human assessment. To overcome the need for human experts it is necessary to develop automated tools for automatically assessing reflectance confocal microscopy imagery. This work presents a novel approach to this task, using a bag of visual words approach to represent and classify en-face optical sections from four distinct strata of the skin. A dictionary of representative features is learned from whitened and normalised patches using hierarchical spherical k-means. Each image is then represented by extracting a dense array of patches and encoding each with the most similar element in the dictionary. Linear discriminant analysis is used as a simple linear classifier. The proposed framework was tested on 308 depth stacks from 54 volunteers. Parameters are tuned using 10 fold cross validation on a training sub-set of the data, and final evaluation was performed on a held out test set. The proposed method generated physically plausible profiles of the distinct strata of human skin, and correctly classified 81.4% of sections in the test set.

  15. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    PubMed Central

    Gribble, Megan; Pertsov, Arkady M.; Shi, Pengcheng

    2013-01-01

    Embryonic heart morphogenesis (EHM) is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling. PMID:24454530

  16. Combined FLIM and reflectance confocal microscopy for epithelial imaging

    NASA Astrophysics Data System (ADS)

    Jabbour, Joey M.; Cheng, Shuna; Shrestha, Sebina; Malik, Bilal; Jo, Javier A.; Applegate, Brian; Maitland, Kristen C.

    2012-03-01

    Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12 mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can potentially serve as a guide in early oral cancer detection..

  17. Use of confocal microscopy for nanoparticle drug delivery through skin

    NASA Astrophysics Data System (ADS)

    Zhang, Leshuai W.; Monteiro-Riviere, Nancy A.

    2013-06-01

    Confocal laser scanning microscopy (CLSM) is a well-used microscopic tool that provides valuable morphological and functional information within cells and tissues. The application of CLSM to skin and the topical penetration of nanoparticles (NP) will be addressed. First, we describe the advantages of confocal microscopy compared to other techniques and its use relative to skin research. Second, we discuss the ability of CLSM to detect single NP. Regarding their interaction with skin, the appropriate method to retain nanoparticle localization in the tissue with minimal fixation is critically important. Also, the interaction of several different types of NP (quantum dots, fullerene and dendrimers) and their interaction with skin detected by CLSM under various conditions (flexed, tape stripped and abraded skin) is reviewed. Finally, human epidermal keratinocytes and dendritic cells that serve as appropriate in vitro models for skin cell interactions and cellular uptake of NP are also discussed. In conclusion, the unique functions of CLSM such as the ability to detect fluorescence, optical sectioning, three dimensional remodeling, as well as its use in the reflection mode in tandem with other methods, provides great promise with broad applications regarding the interactions of nanomaterials with skin.

  18. Template-driven segmentation of confocal microscopy images.

    PubMed

    Chen, Ying-Cheng; Chen, Yung-Chang; Chiang, Ann-Shyn

    2008-03-01

    High quality 3D visualization of anatomic structures is necessary for many applications. The anatomic structures first need to be segmented. A variety of segmentation algorithms have been developed for this purpose. For confocal microscopy images, the noise introduced during the specimen preparation process, such as the procedure of penetration or staining, may cause images to be of low contrast in some regions. This property will make segmentation difficult. Also, the segmented structures may have rugged surfaces in 3D visualization. In this paper, we present a hybrid method that is suitable for segmentation of confocal microscopy images. A rough segmentation result is obtained from the atlas-based segmentation via affine registration. The boundaries of the segmentation result are close to the object boundaries, and are regarded as the initial contours of the active contour models. After convergence of the snake algorithm, the resulting contours in regions of low contrast are locally refined by parametric bicubic surfaces to alleviate the problem of incorrect convergence. The proposed method increases the accuracy of the snake algorithm because of better initial contours. Besides, it can provide smoother segmented results in 3D visualization. PMID:18178286

  19. Precise colloids with tunable interactions for confocal microscopy

    PubMed Central

    Kodger, Thomas E.; Guerra, Rodrigo E.; Sprakel, Joris

    2015-01-01

    Model colloidal systems studied with confocal microscopy have led to numerous insights into the physics of condensed matter. Though confocal microscopy is an extremely powerful tool, it requires a careful choice and preparation of the colloid. Uncontrolled or unknown variations in the size, density, and composition of the individual particles and interactions between particles, often influenced by the synthetic route taken to form them, lead to difficulties in interpreting the behavior of the dispersion. Here we describe the straightforward synthesis of copolymer particles which can be refractive index- and density-matched simultaneously to a non-plasticizing mixture of high dielectric solvents. The interactions between particles are accurately tuned by surface grafting of polymer brushes using Atom Transfer Radical Polymerization (ATRP), from hard-sphere-like to long-ranged electrostatic repulsion or mixed charge attraction. We also modify the buoyant density of the particles by altering the copolymer ratio while maintaining their refractive index match to the suspending solution resulting in well controlled sedimentation. The tunability of the inter-particle interactions, the low volatility of the solvents, and the capacity to simultaneously match both the refractive index and density of the particles to the fluid opens up new possibilities for exploring the physics of colloidal systems. PMID:26420044

  20. Measurement of steep edges and undercuts in confocal microscopy.

    PubMed

    Mueller, T; Jordan, M; Schneider, T; Poesch, A; Reithmeier, E

    2016-05-01

    Confocal microscopy is widely used to measure the surface topography of specimen with a precision in the micrometer range. The measurement uncertainty and quality of the acquired data of confocal microscopy depends on various effects, such as optical aberrations, vibrations of the measurement setup and variations in the surface reflectivity. In this article, the influence of steep edges and undercuts on measurement results is examined. Steep edges on the specimen's surface lead to a reduced detector signal which influences the measurement accuracy and undercuts cause surface regions, which cannot be captured in a measurement. The article describes a method to overcome the negative effects of steep edges and undercuts by capturing several measurements of the surface with different angles between the surface and the optical axis of the objective. An algorithm is introduced which stitches different angle measurements together without knowledge of the exact position and orientation of the rotation axis. Thus, the measurement uncertainty due to steep edges and undercuts can be avoided without expensive high-precision rotation stages and time consuming adjustment of the measurement setup. PMID:27011256

  1. Atomic force microscopy and confocal laser scanning microscopy on the cytoskeleton of permeabilised and embedded cells.

    PubMed

    Meller, Karl; Theiss, Carsten

    2006-03-01

    We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 degrees C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton. PMID:16360280

  2. Adaptive optics in digital micromirror based confocal microscopy

    NASA Astrophysics Data System (ADS)

    Pozzi, P.; Wilding, D.; Soloviev, O.; Vdovin, G.; Verhaegen, M.

    2016-03-01

    This proceeding reports early results in the development of a new technique for adaptive optics in confocal microscopy. The term adaptive optics refers to the branch of optics in which an active element in the optical system is used to correct inhomogeneities in the media through which light propagates. In its most classical form, mostly used in astronomical imaging, adaptive optics is achieved through a closed loop in which the actuators of a deformable mirror are driven by a wavefront sensor. This approach is severely limited in fluorescence microscopy, as the use of a wavefront sensor requires the presence of a bright, point like source in the field of view, a condition rarely satisfied in microscopy samples. Previously reported approaches to adaptive optics in fluorescence microscopy are therefore limited to the inclusion of fluorescent microspheres in the sample, to use as bright stars for wavefront sensors, or time consuming sensorless optimization procedures, requiring several seconds of optimization before the acquisition of a single image. We propose an alternative approach to the problem, implementing sensorless adaptive optics in a Programmable array microscope. A programmable array microscope is a microscope based on a digital micromirror device, in which the single elements of the micromirror act both as point sources and pinholes.

  3. 3D imaging of neutron tracks using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

  4. Cosmetic assessment of the human hair by confocal microscopy.

    PubMed

    Hadjur, Christophe; Daty, Gérard; Madry, Geneviève; Corcuff, Pierre

    2002-01-01

    The optical sectioning property of the confocal microscope offers a breakthrough from the classic observation of the hair in a scanning electron microscope (SEM). Confocal microscopy requires minimal sampling preparation, and the hair can be observed in its natural environment with less damage than by other microscopic methods such as SEM. While used in the reflection mode, the true morphology of the cuticle and the various exogenous deposits at the surface can be identified and quantified. This relatively noninvasive, nondestructive technique is routinely used by us to monitor the efficiency of cleansing shampoos, to assess the homogeneity of layering polymers, and to evaluate the changes they induce in the optical properties of the hair surface in terms of opacity, transparency, and brilliancy. A second important field of investigation uses the fluorescence channel which reveals the internal structure of the hair. Fluorescent probes (rhodamine and its derivatives) demonstrate the routes of penetration and outline the geometry of cortical cells and of the medulla according to their lipophilic or hydrophilic properties. A volume rendering of a hair cylinder provides a better understanding of the interrelationships between cuticle cells, cortical cells, and the medullar channel. This recent technology is becoming an invaluable tool for the cosmetic assessment of the hair. PMID:11998902

  5. Reflectance confocal microscopy for cutaneous infections and infestations.

    PubMed

    Cinotti, E; Perrot, J L; Labeille, B; Cambazard, F

    2016-05-01

    Reflectance confocal microscopy (RCM) is a high-resolution emerging imaging technique that allows non-invasive diagnosis of several cutaneous disorders. A systematic review of the literature on the use of RCM for the study of infections and infestations has been performed to evaluate the current use of this technique and its possible future applications in this field. RCM is particularly suitable for the identification of Sarcoptes scabies, Demodex folliculorum, Ixodes, Dermatophytes and Candida species in the clinical practice and for the follow-up after treatment. The cytopathic effect of herpes simplex virus, varicella zoster virus and molluscipoxvirus is also detectable by this imaging technique even in a pre-vesicular stage. In addition, thanks to its non-invasiveness, RCM allows pathophysiological studies. PMID:26387660

  6. Confocal laser scanning microscopy with spatiotemporal structured illumination.

    PubMed

    Gao, Peng; Nienhaus, G Ulrich

    2016-03-15

    Confocal laser scanning microscopy (CLSM), which is widely utilized in the biological and biomedical sciences, is limited in spatial resolution due to diffraction to about half the light wavelength. Here we have combined structured illumination with CLSM to enhance its spatial resolution. To this end, we have used a spatial light modulator (SLM) to generate fringe patterns of different orientations and phase shifts in the excitation spot without any mechanical movement. We have achieved 1.8 and 1.7 times enhanced lateral and axial resolutions, respectively, by synthesizing the object spectrum along different illumination directions. This technique is thus a promising tool for high-resolution morphological or fluorescence imaging, especially in deep tissue. PMID:26977667

  7. Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

    PubMed

    Lork, Marie; Delvaeye, Mieke; Gonçalves, Amanda; Van Hamme, Evelien; Beyaert, Rudi

    2016-01-01

    Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy. PMID:27613040

  8. Photobleaching property of confocal laser scanning microscopy with masked illumination

    NASA Astrophysics Data System (ADS)

    Kim, DongUk; Moon, Sucbei; Song, Hoseong; Yang, Wenzhong; Kim, Dug Y.

    2010-02-01

    Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e.g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.

  9. Endoscopic probe optics for spectrally encoded confocal microscopy

    PubMed Central

    Kang, DongKyun; Carruth, Robert W.; Kim, Minkyu; Schlachter, Simon C.; Shishkov, Milen; Woods, Kevin; Tabatabaei, Nima; Wu, Tao; Tearney, Guillermo J.

    2013-01-01

    Spectrally encoded confocal microscopy (SECM) is a form of reflectance confocal microscopy that can achieve high imaging speeds using relatively simple probe optics. Previously, the feasibility of conducting large-area SECM imaging of the esophagus in bench top setups has been demonstrated. Challenges remain, however, in translating SECM into a clinically-useable device; the tissue imaging performance should be improved, and the probe size needs to be significantly reduced so that it can fit into luminal organs of interest. In this paper, we report the development of new SECM endoscopic probe optics that addresses these challenges. A custom water-immersion aspheric singlet (NA = 0.5) was developed and used as the objective lens. The water-immersion condition was used to reduce the spherical aberrations and specular reflection from the tissue surface, which enables cellular imaging of the tissue deep below the surface. A custom collimation lens and a small-size grating were used along with the custom aspheric singlet to reduce the probe size. A dual-clad fiber was used to provide both the single- and multi- mode detection modes. The SECM probe optics was made to be 5.85 mm in diameter and 30 mm in length, which is small enough for safe and comfortable endoscopic imaging of the gastrointestinal tract. The lateral resolution was 1.8 and 2.3 µm for the single- and multi- mode detection modes, respectively, and the axial resolution 11 and 17 µm. SECM images of the swine esophageal tissue demonstrated the capability of this device to enable the visualization of characteristic cellular structural features, including basal cell nuclei and papillae, down to the imaging depth of 260 µm. These results suggest that the new SECM endoscopic probe optics will be useful for imaging large areas of the esophagus at the cellular scale in vivo. PMID:24156054

  10. Confocal laser scanning microscopy in study of bone calcification

    NASA Astrophysics Data System (ADS)

    Nishikawa, Tetsunari; Kokubu, Mayu; Kato, Hirohito; Imai, Koichi; Tanaka, Akio

    2012-12-01

    Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15 μm/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

  11. Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nehal, Kishwer S.; Rajadhyaksha, Milind

    2016-02-01

    Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective About 350 publications worldwide have reported the ability of reflectance confocal microscopy (RCM) imaging to detect melanocytic skin lesions in vivo with specificity of 84-88% and sensitivity of 71-92%, and non-melanocytic skin lesions with specificity of 85-97% and sensitivity 100-92%. Lentigo maligna melanoma can be detected with sensitivity of 93% and specificity 82%. While the sensitivity is comparable to that of dermoscopy, the specificity is 2X superior, especially for lightly- and non-pigmented lesions. Dermoscopy combined with RCM imaging is proving to be both highly sensitive and highly specific. Recent studies have reported that the ratio of equivocal (i.e., would have been biopsied) lesions to detected melanomas dropped by ~2X when guided by dermoscopy and RCM imaging, compared to that with dermoscopy alone. Dermoscopy combined with RCM imaging is now being implemented to guide noninvasive diagnosis (to rule out malignancy and biopsy) and to also guide treatment, with promising initial impact: thus far, about 3,000 patients have been saved from biopsies of benign lesions. These are currently under follow-up monitoring. With fluorescence confocal microscopy (FCM) mosaicing, residual basal cell carcinomas can be detected in Mohs surgically excised fresh tissue ex vivo, with sensitivity of 94-97% and specificity 89-94%. FCM mosaicing is now being implemented for guiding Mohs surgery. To date, about 600 Mohs procedures have been performed, guided with mosaicing, and with pathology being performed in parallel to confirm the final outcome. These latest advances demonstrate the promising ability of RCM and FCM to guide patient care.

  12. Mosaic acquisition and processing for optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Shao, Peng; Shi, Wei; Chee, Ryan K. W.; Zemp, Roger J.

    2012-08-01

    In optical-resolution photo-acoustic microscopy (OR-PAM), data acquisition time is limited by both laser pulse repetition rate (PRR) and scanning speed. Optical-scanning offers high speed, but limited, field of view determined by ultrasound transducer sensitivity. In this paper, we propose a hybrid optical and mechanical-scanning OR-PAM system with mosaic data acquisition and processing. The system employs fast-scanning mirrors and a diode-pumped, nanosecond-pulsed, Ytterbium-doped, 532-nm fiber laser with PRR up to 600 kHz. Data from a sequence of image mosaic patches is acquired systematically, at predetermined mechanical scanning locations, with optical scanning. After all imaging locations are covered, a large panoramic scene is generated by stitching the mosaic patches together. Our proposed system is proven to be at least 20 times faster than previous reported OR-PAM systems.

  13. Diffusion of photoacid generators by laser scanning confocal microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Ping L.; Webber, Stephen E.; Mendenhall, J.; Byers, Jeffrey D.; Chao, Keith K.

    1998-06-01

    Diffusion of the photogenerated acid during the period of time between exposure and development can cause contrast loss and ultimately loss of the latent image. This is especially relevant for chemically amplified photoresists that require a post-exposure baking step, which in turn facilitates acid diffusion due to the high temperature normally employed. It is thus important to develop techniques with good spatial resolution to monitor the photogeneration of acid. More precisely, we need techniques that provide two distinct types of information: spatial resolution on various length scales within the surface layer and also sufficient depth resolution so that one can observe the transition from very surface layer to bulk structure in the polymer blend coated on silicon substrate. Herein laser scanning confocal microscopy is used to evaluate the resist for the first time. We report the use of the confocal microscopy to map the pag/dye distribution in PHS matrices, with both reflectance images and fluorescence images. A laser beam is focused onto a small 3D volume element, termed a voxel. It is typically 200 nm X 200 nm laterally and 800 nm axially. The illuminated voxel is viewed such that only signals emanating from this voxel are detected, i.e., signal from outside the probed voxel is not detected. By adjusting the vertical position of the laser focal point, the voxel can be moved to the designated lateral plane to produce an image. Contrast caused by topology difference between the exposed and unexposed area can be eliminated. Bis-p-butylphenyl iodonium triflat (7% of polyhydroxystyrene) is used as photoacid generators. 5% - 18% (by weight, PHS Mn equals 13 k) resist in PGMEA solution is spin cast onto the treated quartz disk with thickness of 1.4 micrometers , 5 micrometers space/10 micrometers pitch chrome mask is used to generate the pattern with mercury DUV illumination. Fluoresceinamine, the pH-sensitive dye, is also used to enhance the contrast of

  14. Managing multiple image stacks from confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Zerbe, Joerg; Goetze, Christian H.; Zuschratter, Werner

    1999-05-01

    A major goal in neuroanatomy is to obtain precise information about the functional organization of neuronal assemblies and their interconnections. Therefore, the analysis of histological sections frequently requires high resolution images in combination with an overview about the structure. To overcome this conflict we have previously introduced a software for the automatic acquisition of multiple image stacks (3D-MISA) in confocal laser scanning microscopy. Here, we describe a Windows NT based software for fast and easy navigation through the multiple images stacks (MIS-browser), the visualization of individual channels and layers and the selection of user defined subregions. In addition, the MIS browser provides useful tools for the visualization and evaluation of the datavolume, as for instance brightness and contrast corrections of individual layers and channels. Moreover, it includes a maximum intensity projection, panning and zoom in/out functions within selected channels or focal planes (x/y) and tracking along the z-axis. The import module accepts any tiff-format and reconstructs the original image arrangement after the user has defined the sequence of images in x/y and z and the number of channels. The implemented export module allows storage of user defined subregions (new single image stacks) for further 3D-reconstruction and evaluation.

  15. Rhinosporidium seeberi Nuclear Cycle Activities Using Confocal Microscopy.

    PubMed

    Delfino, Darly; Mendoza, Leonel; Vilela, Raquel

    2016-02-01

    Rhinosporidium seeberi is an uncultivated Ichthyosporean infecting animals, including humans. Recent studies suggested R. seeberi undergoes synchronized nuclear division without cytokinesis. We used confocal microscopy to investigate R. seeberi nuclear division cycles in formalin-fixed tissues stained with DAPI and phalloidin. We report that R. seeberi nuclei in juvenile and intermediary sporangia synchronously divided without cytokinesis. Intermediary sporangia display numerous 3-4 μm nuclei at different mitotic stages as well as a thick inner layer with strong affinity for phalloidin. Mature sporangia showed numerous 5-12 μm cell-walled endospores, each containing a 2-4 μm in diameter nucleus. Phalloidin did not bind to the inner layers of mature sporangia or endospores. The development of a "germinative zone" in the inner layer of mature sporangia containing hundreds of nuclei was also confirmed. This study establishes that during the R. seeberi life cycle synchronous nuclear divisions without cytokinesis takes place, resulting in the formation of thousands of nuclei. Cytokinesis, on the other hand, is a 1-time event and occurs in the latest stages of intermediate sporangia, after the formation of thousands of nuclei and just before mature sporangia development. PMID:26461427

  16. Improving transverse resolution of confocal microscopy through spatiotemporal modulation

    NASA Astrophysics Data System (ADS)

    Wang, Baokai; Zou, Limin; Zhang, Su; Tan, Jiubin

    2015-11-01

    A new method is proposed in this paper to improve transverse resolution of a confocal microscope. By setting up the model of a confocal microscope system through spatiotemporal modulation with moving gratings or acousto-optical modulation without defocus distance under coherent light illumination and deducing two-dimensional coherent image formula and transfer function, simulation tests are run with or without spatiotemporal modulation to prove the effectiveness of the proposed method. Simulation results indicate the proposed method can be used to improve the transverse resolution of a confocal microscope system.

  17. Laser ablation of basal cell carcinomas guided by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Sierra, Heidy; Cordova, Miguel; Nehal, Kishwer; Rossi, Anthony; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

    2016-02-01

    Laser ablation offers precise and fast removal of superficial and early nodular types of basal cell carcinomas (BCCs). Nevertheless, the lack of histological confirmation has been a limitation. Reflectance confocal microscopy (RCM) imaging combined with a contrast agent can offer cellular-level histology-like feedback to detect the presence (or absence) of residual BCC directly on the patient. We conducted an ex vivo bench-top study to provide a set of effective ablation parameters (fluence, number of passes) to remove superficial BCCs while also controlling thermal coagulation post-ablation to allow uptake of contrast agent. The results for an Er:YAG laser (2.9 um and pulse duration 250us) show that with 6 passes of 25 J/cm2, thermal coagulation can be effectively controlled, to allow both the uptake of acetic acid (contrast agent) and detection of residual (or absence) BCCs. Confirmation was provided with histological examination. An initial in vivo study on 35 patients shows that the uptake of contrast agent aluminum chloride) and imaging quality is similar to that observed in the ex vivo study. The detection of the presence of residual tumor or complete clearance was confirmed in 10 wounds with (additional) histology and in 25 lesions with follow-up imaging. Our results indicate that resolution is sufficient but further development and use of appropriate contrast agent are necessary to improve sensitivity and specificity. Advances in RCM technology for imaging of lateral and deep margins directly on the patient may provide less invasive, faster and less expensive image-guided approaches for treatment of BCCs.

  18. Measuring Corneal Haze by Using Scheimpflug Photography and Confocal Microscopy

    PubMed Central

    McLaren, Jay W.; Wacker, Katrin; Kane, Katrina M.; Patel, Sanjay V.

    2016-01-01

    Purpose We compared corneal backscatter estimated from a Scheimpflug camera with backscatter estimated from a clinical confocal microscope across a wide range of corneal haze. Methods A total of 59 corneas from 35 patients with a range of severity of Fuchs' endothelial corneal dystrophy and 15 corneas from 9 normal participants were examined using a Scheimpflug camera (Pentacam) and a confocal microscope (ConfoScan 4). The mean image brightness from the anterior 120 μm, midcornea, and posterior 60 μm of the cornea across the central 2 mm recorded by the Scheimpflug camera and analogous regions from the confocal microscope were measured and standardized. Differences between instruments and correlations between backscatter and disease severity were determined by using generalized estimating equation models. Results Backscatter measured by the two instruments in the anterior and midcornea were correlated (r = 0.67 and 0.43, respectively, P < 0.001), although in the posterior cornea they were not correlated (r = 0.13, P = 0.66). Measured with the Scheimpflug camera, mean backscatter from the anterior and midcornea were greater, whereas backscatter from the posterior cornea was lower (P < 0.001) than that measured by the confocal microscope. Backscatter from the anterior cornea was correlated with disease severity for both instruments (Scheimpflug, r = 0.55, P < 0.001; confocal, r = 0.49, P = 0.003). Conclusions The Scheimpflug camera and confocal microscope should not be used interchangeably to measure corneal haze. The ability to detect changes in backscatter with disease severity is superior with the Scheimpflug camera. However, the confocal microscope provides higher resolution of corneal structure. PMID:26803798

  19. Progress in reflectance confocal microscopy for imaging oral tissues in vivo

    NASA Astrophysics Data System (ADS)

    Peterson, Gary; Zanoni, Daniella K.; Migliacci, Jocelyn; Cordova, Miguel; Rajadhyaksha, Milind; Patel, Snehal

    2016-02-01

    We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and "video-mosaicing", extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

  20. In vivo confocal microscopy of meibomian glands in primary blepharospasm

    PubMed Central

    Lin, Tong; Gong, Lan

    2016-01-01

    Abstract The aim of the study was to evaluate the morphological changes of meibomian glands (MGs) in primary blepharospasm (PBS) by in vivo laser scanning confocal microscopy (LSCM) and to investigate the correlations between clinical data of PBS and LSCM parameters of MGs. This prospective and case–control study recruited 30 consecutive PBS patients and 30 age- and gender-matched healthy controls. After questionnaire assessments of ocular surface disease index (OSDI), Jankovic rating scale, and blepharospasm disability index, all subjects underwent blink rate evaluation, tear film break-up time (TBUT), corneal fluorescein staining (CFS), Schirmer test, MG expressibility, meibum quality, MG dropout, and LSCM examination of the MGs. The main LSCM outcomes included the mean MG acinar area and density, orifice diameter, meibum secretion reflectivity, acinar irregularity, and inhomogeneity of interstice and acinar wall. The PBS patients had significantly higher blink rate, higher OSDI and CFS scores, lower TBUT and Schirmer test value, and worse MG expressibility than the controls (All P < 0.05), whereas meibum quality showed no difference (P > 0.05). The PBS patients showed lower values of MG acinar area, orifice diameter and meibum secretion reflectivity, and higher scores of acinar irregularity and inhomogeneity of interstices than the controls (All P < 0.05). For the PBS patients, the severity of blepharospasm evaluated by JCR scale was strong correlated with MG acinar area (P < 0.001), orifice diameter (P = 0.002), meibum secretion reflectivity (P = 0.002), and MG acinar irregularity (P = 0.013). The MG expressibility was significantly correlated to MG acinar area (P = 0.039), orifice diameter (P < 0.001), and MG acinar irregularity (P = 0.014). The OSDI score was moderate correlated with MG acinar irregularity (P = 0.016), whereas the TBUT value was positively correlated with MG acinar area (P = 0.045) and negatively correlated to MG acinar

  1. Confocal Microscopy of Jammed Matter: From Elasticity to Granular Thermodynamics

    NASA Astrophysics Data System (ADS)

    Jorjadze, Ivane

    Packings of particles are ubiquitous in nature and are of interest not only to the scientific community but also to the food, pharmaceutical, and oil industries. In this thesis we use confocal microscopy to investigate packing geometry and stress transmission in 3D jammed particulate systems. By introducing weak depletion attraction we probe the accessible phase-space and demonstrate that a microscopic approach to jammed matter gives validity to statistical mechanics framework, which is intriguing because our particles are not thermally activated. We show that the fluctuations of the local packing parameters can be successfully captured by the recently proposed 'granocentric' model, which generates packing statistics according to simple stochastic processes. This model enables us to calculate packing entropy and granular temperature, the so-called 'compactivity', therefore, providing a basis for a statistical mechanics of granular matter. At a jamming transition point at which there are formed just enough number of contacts to guarantee the mechanical stability, theoretical arguments suggest a singularity which gives rise to the surprising scaling behavior of the elastic moduli and the microstructure, as observed in numerical simulations. Since the contact network in 3D is typically hidden from view, experimental test of the scaling law between the coordination number and the applied pressure is lacking in the literature. Our data show corrections to the linear scaling of the pressure with density which takes into account the creation of contacts. Numerical studies of vibrational spectra, in turn, reveal sudden features such as excess of low frequency modes, dependence of mode localization and structure on the pressure. Chapter four describes the first calculation of vibrational density of states from the experimental 3D data and is in qualitative agreement with the analogous computer simulations. We study the configurational role of the pressure and demonstrate

  2. Reflectance confocal microscopy of red blood cells: simulation and experiment

    PubMed Central

    Zeidan, Adel; Yelin, Dvir

    2015-01-01

    Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient’s health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology. PMID:26600999

  3. High-resolution confocal Raman microscopy using pixel reassignment.

    PubMed

    Roider, Clemens; Ritsch-Marte, Monika; Jesacher, Alexander

    2016-08-15

    We present a practical modification of fiber-coupled confocal Raman scanning microscopes that is able to provide high confocal resolution in conjunction with high light collection efficiency. For this purpose, the single detection fiber is replaced by a hexagonal lenslet array in combination with a hexagonally packed round-to-linear multimode fiber bundle. A multiline detector is used to collect individual Raman spectra for each fiber. Data post-processing based on pixel reassignment allows one to improve the lateral resolution by up to 41% compared to a single fiber of equal light collection efficiency. We present results from an experimental implementation featuring seven collection fibers, yielding a resolution improvement of about 30%. We believe that our implementation represents an attractive upgrade for existing confocal Raman microscopes that employ multi-line detectors. PMID:27519099

  4. FTIR microscopy and confocal Raman microscopy for studying lateral drug diffusion from a semisolid formulation.

    PubMed

    Gotter, B; Faubel, W; Neubert, R H H

    2010-01-01

    Fourier transform infrared (FTIR) microscopy was applied to obtain information on lateral drug diffusion of dithranol in artificial acceptor membranes. Lateral (2D) drug distribution into an artificial membrane was investigated on an area of 300microm x 1000microm with a lateral resolution of 25microm x 25microm by integrating a specific IR band located at 1430cm(-1). The concentration profiles show a heterogeneous distribution of dithranol particles resulting in non-uniform drug diffusion. Use of the FTIR microscope either in the transmission or in the reflection mode was restricted to a thickness of the DDC membrane <15microm. The third dimension (depth profile) was analysed by means of confocal Raman microscopy (CRM). In an artificial membrane, the depth range from a minimum of 1.5microm up to a maximum of 49microm was analysed for dithranol distribution. PMID:19615444

  5. Spectral confocal reflection microscopy using a white light source

    NASA Astrophysics Data System (ADS)

    Booth, M.; Juškaitis, R.; Wilson, T.

    2008-08-01

    We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

  6. Confocal Raman microscopy for identification of bacterial species in biofilms

    NASA Astrophysics Data System (ADS)

    Beier, Brooke D.; Quivey, Robert G.; Berger, Andrew J.

    2011-03-01

    Implemented through a confocal microscope, Raman spectroscopy has been used to distinguish between biofilm samples of two common oral bacteria species, Streptococcus sanguinis and mutans, which are associated with healthy and cariogenic plaque, respectively. Biofilms of these species are studied as a model of dental plaque. A prediction model has been calibrated and validated using pure biofilms. This model has been used to identify the species of transferred and dehydrated samples (much like a plaque scraping) as well as hydrated biofilms in situ. Preliminary results of confocal Raman mapping of species in an intact two-species biofilm will be shown.

  7. Full-field interferometric confocal microscopy using a VCSEL array

    PubMed Central

    Redding, Brandon; Bromberg, Yaron; Choma, Michael A.; Cao, Hui

    2014-01-01

    We present an interferometric confocal microscope using an array of 1200 VCSELs coupled to a multimode fiber. Spatial coherence gating provides ~18,000 continuous virtual pinholes allowing an entire en face plane to be imaged in a snapshot. This approach maintains the same optical sectioning as a scanning confocal microscope without moving parts, while the high power of the VCSEL array (~5 mW per laser) enables high-speed image acquisition with integration times as short as 100 µs. Interferometric detection also recovers the phase of the image, enabling quantitative phase measurements and improving the contrast when imaging phase objects. PMID:25078199

  8. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Hayashi, Shinichi

    2016-08-01

    Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy with a 2-fold higher lateral resolution than conventional wide-field fluorescence (WF) microscopy. Confocal fluorescence (CF) microscopy has approximately the same optical cutoff frequency as SIM; however, the maximum theoretical increase in lateral resolution over that of WF is 1.4-fold with an infinitesimal pinhole diameter. Quantitative comparisons based on an analytical imaging formula revealed that modulation transfer functions (MTFs) of SIM reconstructed images before postprocessing are nearly identical to those of CF images recorded with an infinitesimal pinhole diameter. Here, we propose a new method using an adequate pinhole diameter combined with the use of an apodized Fourier inverse filter to increase the lateral resolution of CF images to as much as that SIM images without significant noise degradation in practice. Furthermore, the proposed method does not require a posteriori parameterization and has reproducibility. This approach can be easily applied to conventional laser scanning CF, spinning disk CF, and multiphoton microscopies.

  9. WHOLE INSECT AND MAMMALIAN EMBRYO IMAGING WITH CONFOCAL MICROSCOPY: MORPHOLOGY AND APOPTOSIS

    EPA Science Inventory

    Background: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates...

  10. Ex Vivo (Fluorescence) Confocal Microscopy in Surgical Pathology: State of the Art.

    PubMed

    Ragazzi, Moira; Longo, Caterina; Piana, Simonetta

    2016-05-01

    First developed in 1957, confocal microscopy is a powerful imaging tool that can be used to obtain near real-time reflected light images of untreated human tissue with nearly histologic resolution. Besides its research applications, in the last decades, confocal microscopy technology has been proposed as a useful device to improve clinical diagnosis, especially in ophthalmology, dermatology, and endomicroscopy settings, thanks to advances in instrument development. Compared with the wider use of the in vivo tissue assessment, ex vivo applications of confocal microscopy are not fully explored. A comprehensive review of the current literature was performed here, focusing on the reliable applications of ex vivo confocal microscopy in surgical pathology and on some potential evolutions of this new technique from pathologists' viewpoint. PMID:27058244

  11. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR CALIBRATION, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of emitted signals. The accuracy of these measurements demands that...

  12. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: SPECTROSCOPY AND FOUNDATIONS FOR QUANTITATION

    EPA Science Inventory

    The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For man...

  13. Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

    PubMed

    Paddock, Stephen W; Eliceiri, Kevin W

    2014-01-01

    Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques. PMID:24052346

  14. The use of reflectance confocal microscopy in selected inflammatory skin diseases.

    PubMed

    Białek-Galas, Kamila; Wielowieyska-Szybińska, Dorota; Dyduch, Grzegorz; Wojas-Pelc, Anna

    2015-06-01

    Reflectance confocal microscopy is a modern, non-invasive diagnostic method that enables real-time imaging of the epidermis and upper layers of the dermis with nearly histological precision and high contrast. The application of this technology to skin imaging during the last years has resulted in progress of dermatological diagnosis, providing virtual access to living skin, without the need for conventional histopathology. The presented method potentially has broad application in the diagnosis of skin diseases. This article provides a summary of the latest reports and previous achievements in the field of reflectance confocal microscopy. General characteristics of confocal images in selected inflammatory skin diseases are presented. PMID:26247522

  15. Cytosolic pH gradients in cultured neuronal cell lines studied by laser scanning confocal microscopy, real-time confocal microscopy, and spectral imaging microscopy

    NASA Astrophysics Data System (ADS)

    Sanchez-Armass, Sergio; Sennoune, Souad; Martinez, Gloria M.; Ortega, Filiberta; Martinez-Zaguilan, Raul

    2002-06-01

    Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.

  16. Anti-translational research: from the bedside back to the bench for reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gareau, Daniel

    2014-03-01

    The reflectance confocal microscope has made translational progress in dermatology. 0.5 micrometer lateral resolution, 0.75mm field-of-view and excellent temporal resolution at ~15 frames/second serve the VivaScope well in the clinic, but it may be overlooked in basic research. This work reviews high spatiotemporal confocal microscopy and presents images acquired of various samples: zebra fish embryo where melanocytes with excellent contrast overly the spinal column, chicken embryo, where myocardium is seen moving at 15 frames/ second, calcium spikes in dendrites (fluorescence mode) just beyond the temporal resolution, and human skin where blood cells race through the artereovenous microvasculature. For an introduction to confocal microscopy, see: http://dangareau.net.s69818.gridserver.com/science/confocal-microscopy

  17. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

    PubMed Central

    Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H.; Wouters, Fred S.; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

    2013-01-01

    We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions. PMID:24324140

  18. Comparison between optical-resolution photoacoustic microscopy and confocal laser scanning microscopy for turbid sample imaging

    NASA Astrophysics Data System (ADS)

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

    Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering.

  19. Comparison between optical-resolution photoacoustic microscopy and confocal laser scanning microscopy for turbid sample imaging.

    PubMed

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

    Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering. PMID:26256640

  20. Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy

    PubMed Central

    Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.

    2013-01-01

    In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

  1. In-vivo multi-spectral confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rouse, Andrew R.; Udovich, Joshua A.; Gmitro, Arthur F.

    2005-03-01

    A multi-spectral confocal microendoscope (MCME) for in-vivo imaging has been developed. The MCME employs a flexible fiber-optic catheter coupled to a slit-scan confocal microscope with an imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The focus mechanism allows for imaging to a maximum tissue depth of 200 microns. 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 3 micron lateral resolution and 30 micron axial resolution. The system incorporates two laser sources and is therefore capable of simultaneous acquisition of spectra from multiple dyes using dual excitation. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 8nm to 16nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersion characteristics 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. In-vitro, and ex-vivo multi-spectral results are presented.

  2. Enhanced confocal microscopy and ophthalmoscopy with polarization imaging

    NASA Astrophysics Data System (ADS)

    Campbell, Melanie C. W.; Bueno, Juan M.; Cookson, Christopher J.; Liang, Qingyuan; Kisilak, Marsha L.; Hunter, Jennifer J.

    2005-09-01

    We previously developed a Mueller matrix formalism to improve confocal imaging in microscopes and ophthalmoscopes. Here we describe a procedure simplified by firstly introducing a generator of polarization states in the illumination pathway of a confocal scanning laser microscope and secondly computing just four elements of the Mueller matrix of any sample and instrument combination. Using a subset of Mueller matrix elements, the best images are reconstructed. The method was tested for samples with differing properties (specular, diffuse and partially depolarizing). Images were also studied of features at the rear of the eye. The best images obtained with this technique were compared to the original images and those obtained from frame averaging. Images corresponding to non-polarized incident light were also computed. For all cases, the best reconstructed images were of better quality than both the original and frame-averaged images. The best reconstructed images also showed an improvement compared with the images corresponding to non polarized light. This methodology will have broad application in biomedical imaging.

  3. Variational attenuation correction in two-view confocal microscopy

    PubMed Central

    2013-01-01

    Background Absorption and refraction induced signal attenuation can seriously hinder the extraction of quantitative information from confocal microscopic data. This signal attenuation can be estimated and corrected by algorithms that use physical image formation models. Especially in thick heterogeneous samples, current single view based models are unable to solve the underdetermined problem of estimating the attenuation-free intensities. Results We present a variational approach to estimate both, the real intensities and the spatially variant attenuation from two views of the same sample from opposite sides. Assuming noise-free measurements throughout the whole volume and pure absorption, this would in theory allow a perfect reconstruction without further assumptions. To cope with real world data, our approach respects photon noise, estimates apparent bleaching between the two recordings, and constrains the attenuation field to be smooth and sparse to avoid spurious attenuation estimates in regions lacking valid measurements. Conclusions We quantify the reconstruction quality on simulated data and compare it to the state-of-the art two-view approach and commonly used one-factor-per-slice approaches like the exponential decay model. Additionally we show its real-world applicability on model organisms from zoology (zebrafish) and botany (Arabidopsis). The results from these experiments show that the proposed approach improves the quantification of confocal microscopic data of thick specimen. PMID:24350574

  4. Reconstruction and exploration of three-dimensional confocal microscopy data in an immersive virtual environment.

    PubMed

    Ai, Zhuming; Chen, Xue; Rasmussen, Mary; Folberg, Robert

    2005-07-01

    An immersive virtual environment for interactive three-dimensional reconstruction and exploration of confocal microscopy data is presented. For some structures automatic alignment of serial sections can lead to geometric distortions. The superior visual feedback of a Virtual Reality system is used to aid in registering and aligning serial sections interactively. An ImmersaDesk Virtual Reality display system is used for display and interaction with the volumetric confocal data. Detailed methods for handling both single-section and multi-section confocal data are described. PMID:15893451

  5. The use of laser scanning confocal microscopy (LSCM) in materials science.

    PubMed

    Hovis, D B; Heuer, A H

    2010-12-01

    Laser scanning confocal microscopes are essential and ubiquitous tools in the biological, biochemical and biomedical sciences, and play a similar role to scanning electron microscopes in materials science. However, modern laser scanning confocal microscopes have a number of advantages for the study of materials, in addition to their obvious uses for high resolution reflected and transmitted light optical microscopy. In this paper, we provide several examples that exploit the laser scanning confocal microscope's capabilities of pseudo-infinite depth of field imaging, topographic imaging, photo-stimulated luminescence imaging and Raman spectroscopic imaging. PMID:21077878

  6. Digital image acquisition in in vivo confocal microscopy.

    PubMed

    Petroll, W M; Cavanagh, H D; Lemp, M A; Andrews, P M; Jester, J V

    1992-01-01

    A flexible system for the real-time acquisition of in vivo images has been developed. Images are generated using a tandem scanning confocal microscope interfaced to a low-light-level camera. The video signal from the camera is digitized and stored using a Gould image processing system with a real-time digital disk (RTDD). The RTDD can store up to 3200 512 x 512 pixel images at video rates (30 images s-1). Images can be input directly from the camera during the study, or off-line from a Super VHS video recorder. Once a segment of experimental interest is digitized onto the RTDD, the user can interactively step through the images, average stable sequences, and identify candidates for further processing and analysis. Examples of how this system can be used to study the physiology of various organ systems in vivo are presented. PMID:1552573

  7. Group refractive index reconstruction with broadband interferometric confocal microscopy

    PubMed Central

    Marks, Daniel L.; Schlachter, Simon C.; Zysk, Adam M.; Boppart, Stephen A.

    2010-01-01

    We propose a novel method of measuring the group refractive index of biological tissues at the micrometer scale. The technique utilizes a broadband confocal microscope embedded into a Mach–Zehnder interferometer, with which spectral interferograms are measured as the sample is translated through the focus of the beam. The method does not require phase unwrapping and is insensitive to vibrations in the sample and reference arms. High measurement stability is achieved because a single spectral interferogram contains all the information necessary to compute the optical path delay of the beam transmitted through the sample. Included are a physical framework defining the forward problem, linear solutions to the inverse problem, and simulated images of biologically relevant phantoms. PMID:18451922

  8. Inverse image alignment method for image mosaicing and video stabilization in fundus indocyanine green angiography under confocal scanning laser ophthalmoscope.

    PubMed

    Zhou, Yongjin; Xue, Hui; Wan, Mingxi

    2003-01-01

    An efficient image registration algorithm, the Inverse Compositional image alignment method based on minimization of Sum of Squared Differences of images, is applied in fundus blood vessel angiography under confocal scanning laser ophthalmoscope, to build image mosaics which have larger field of view without loss of resolution to assist diagnosis. Furthermore, based on similar technique, the angiography video stabilization algorithm is implemented for fundus documenting. The actual underlying models of motion between images and corresponding convergence criteria are also discussed. The experiment results in fundus images demonstrate the effectiveness of the registration scheme. PMID:14575786

  9. Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

    PubMed

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes. PMID:27515076

  10. Corneal confocal microscopy: Recent progress in the evaluation of diabetic neuropathy

    PubMed Central

    Papanas, Nikolaos; Ziegler, Dan

    2015-01-01

    The present brief review discusses recent progress with corneal confocal microscopy for the evaluation of diabetic sensorimotor polyneuropathy. Corneal confocal microscopy is a new, non-invasive and reproducible diagnostic modality, and it can also be easily applied for patient follow up. It enables new perspectives of studying the natural history of diabetic sensorimotor polyneuropathy, severity of nerve fiber pathology and documenting early nerve fiber regeneration after therapeutic intervention. It shows moderate to high sensitivity and specificity for the timely diagnosis of diabetic sensorimotor polyneuropathy. Currently, corneal confocal microscopy is mainly used in specialized centers, but deserves more widespread application for the assessment of diabetic sensorimotor polyneuropathy. Finally, further progress is required in terms of technical improvements for automated nerve fiber quantification and for analysis of larger images. PMID:26221515

  11. Corneal confocal microscopy: Recent progress in the evaluation of diabetic neuropathy.

    PubMed

    Papanas, Nikolaos; Ziegler, Dan

    2015-07-01

    The present brief review discusses recent progress with corneal confocal microscopy for the evaluation of diabetic sensorimotor polyneuropathy. Corneal confocal microscopy is a new, non-invasive and reproducible diagnostic modality, and it can also be easily applied for patient follow up. It enables new perspectives of studying the natural history of diabetic sensorimotor polyneuropathy, severity of nerve fiber pathology and documenting early nerve fiber regeneration after therapeutic intervention. It shows moderate to high sensitivity and specificity for the timely diagnosis of diabetic sensorimotor polyneuropathy. Currently, corneal confocal microscopy is mainly used in specialized centers, but deserves more widespread application for the assessment of diabetic sensorimotor polyneuropathy. Finally, further progress is required in terms of technical improvements for automated nerve fiber quantification and for analysis of larger images. PMID:26221515

  12. Emulation and design of terahertz reflection-mode confocal scanning microscopy based on virtual pinhole

    NASA Astrophysics Data System (ADS)

    Yang, Yong-fa; Li, Qi

    2014-12-01

    In the practical application of terahertz reflection-mode confocal scanning microscopy, the size of detector pinhole is an important factor that determines the performance of spatial resolution characteristic of the microscopic system. However, the use of physical pinhole brings some inconvenience to the experiment and the adjustment error has a great influence on the experiment result. Through reasonably selecting the parameter of matrix detector virtual pinhole (VPH), it can efficiently approximate the physical pinhole. By using this approach, the difficulty of experimental calibration is reduced significantly. In this article, an imaging scheme of terahertz reflection-mode confocal scanning microscopy that is based on the matrix detector VPH is put forward. The influence of detector pinhole size on the axial resolution of confocal scanning microscopy is emulated and analyzed. Then, the parameter of VPH is emulated when the best axial imaging performance is reached.

  13. Measuring skin penetration by confocal Raman microscopy (CRM): correlation to results from conventional experiments

    NASA Astrophysics Data System (ADS)

    Lunter, Dominique; Daniels, Rolf

    2016-03-01

    Confocal Raman microscopy has become an advancing technique in the characterization of drug transport into the skin. In this study the skin penetration of a local anesthetic from a semisolid preparation was investigated. Furthermore, the effect of the chemical enhancers propylene glycol and POE-23-lauryl ether on its penetration was investigated. The results show that confocal Raman microscopy may provide detailed information on the penetration of APIs into the skin and may elucidate their distribution within the skin with high resolution. The results of the CRM analysis are fully in line with those of conventional permeation and penetration experiments.

  14. Sheet-scanned dual-axis confocal (SS-DAC) microscopy using Richardson-Lucy deconvolution

    PubMed Central

    Wang, Danni; Meza, Daphne; Wang, Yu; Gao, Liang; Liu, Jonathan T.C.

    2015-01-01

    We have previously developed a line-scanned dual-axis confocal (LS-DAC) microscope with subcellular resolution suitable for high-frame-rate diagnostic imaging at shallow depths. Due to the loss of confocality along one dimension, the contrast (signal-to-background ratio) of a LS-DAC microscope is deteriorated compared to a point-scanned DAC microscope. However, by using a sCMOS camera for detection, a short oblique light-sheet is imaged at each scanned position. Therefore, by scanning the light sheet in only one dimension, a thin 3D volume is imaged. Both sequential two-dimensional deconvolution and three-dimensional deconvolution are performed on the thin image volume to improve the resolution and contrast of one en face confocal image section at the center of the volume, a technique we call sheet-scanned dual-axis confocal (SS-DAC) microscopy. PMID:26466290

  15. Sheet-scanned dual-axis confocal microscopy using Richardson-Lucy deconvolution.

    PubMed

    Wang, D; Meza, D; Wang, Y; Gao, L; Liu, J T C

    2014-09-15

    We have previously developed a line-scanned dual-axis confocal (LS-DAC) microscope with subcellular resolution suitable for high-frame-rate diagnostic imaging at shallow depths. Due to the loss of confocality along one dimension, the contrast (signal-to-background ratio) of a LS-DAC microscope is deteriorated compared to a point-scanned DAC microscope. However, by using a sCMOS camera for detection, a short oblique light-sheet is imaged at each scanned position. Therefore, by scanning the light sheet in only one dimension, a thin 3D volume is imaged. Both sequential two-dimensional deconvolution and three-dimensional deconvolution are performed on the thin image volume to improve the resolution and contrast of one en face confocal image section at the center of the volume, a technique we call sheet-scanned dual-axis confocal (SS-DAC) microscopy. PMID:26466290

  16. High-speed confocal fluorescence lifetime imaging microscopy by analog mean-delay method

    NASA Astrophysics Data System (ADS)

    Won, Youngjae; Kim, Donguk; Yang, Wenzhong; Kim, Dug Y.

    2010-02-01

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

  17. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    PubMed Central

    Beltran, Nohra E.; Garcia, Laura E.; Garcia-Lorenzana, Mario

    2013-01-01

    The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine). The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10%) for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (P < 0.01). Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia. PMID:23841094

  18. Reflectance confocal microscopy for scarring and non-scarring alopecia real-time assessment.

    PubMed

    Ardigò, Marco; Agozzino, Marina; Franceschini, Chiara; Donadio, Carlo; Abraham, Leonardo Spagnol; Barbieri, Luca; Sperduti, Isabella; Berardesca, Enzo; González, Salvador

    2016-07-01

    Clinical management of alopecia represents one of the major issues in dermatology. Scalp biopsies are not easily accepted because of the high bleeding and sensitive anatomical area. Trichoscopy is routinely used for diagnosis of alopecia, but in several cases lack to provide sufficient information on the status of the disease. Recently, reflectance confocal microscopy demonstrated its usefulness for the evaluation of several inflammatory skin condition and preliminary reports about alopecia have been proposed in the literature. The aim was to identify the confocal features characterizing scarring and non-scarring alopecia. Reflectance confocal microscopy from 86 patients affected by scarring (28 lichen planopilaris and 9 lupus erythematosus) and non-scarring alopecia (30 androgenic alopecia and 19 alopecia areata), were retrospectively, blinded evaluated. Good concordance between different readers on the confocal criteria has been assessed. Statistical significant features, specific for scarring alopecia and non-scarring alopecia have been identified. In this study, data on reflectance confocal microscopy features useful for the differential diagnosis between scarring and non-scarring alopecia have been identified. Further studies focusing on the use of this non-invasive technique in the therapeutic follow-up and distinction of sub-entities of alopecia are still required. PMID:27225248

  19. Clinical applications of in vivo fluorescence confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

    2008-02-01

    Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In

  20. Effects of Fluorescein Staining on Laser In Vivo Confocal Microscopy Images of the Cornea

    PubMed Central

    Sindt, Christine W.; Critser, D. Brice; Grout, Trudy K.; Kern, Jami R.

    2012-01-01

    This study was designed to identify whether topical fluorescein, a common ophthalmic tool, affects laser in vivo confocal microscopy of the cornea, a tool with growing applications. Twenty-five eye care specialists were asked to identify presence or absence of fluorescein in 99 confocal micrographs of healthy corneas. Responses were statistically similar to guessing for the epithelium (48% ± 14% of respondents correct per image) and the subbasal nerve plexus (49% ± 11% correct), but results were less clear for the stroma. Dendritic immune cells were quantified in bilateral images from subjects who had been unilaterally stained with fluorescein. Density of dendritic immune cells was statistically similar between the unstained and contralateral stained eyes of 24 contact lens wearers (P = .72) and of 10 nonwearers (P = .53). Overall, the results indicated that fluorescein staining did not interfere with laser confocal microscopy of corneal epithelium, subbasal nerves, or dendritic immune cells. PMID:22363837

  1. Determination of Nanogram Microparticles from Explosives after Real Open-Air Explosions by Confocal Raman Microscopy.

    PubMed

    Zapata, Félix; García-Ruiz, Carmen

    2016-07-01

    Explosives are increasingly being used for terrorist attacks to cause devastating explosions. The detection of their postblast residues after an explosion is a high challenge, which has been barely investigated, particularly using spectroscopic techniques. In this research, a novel methodology using confocal Raman microscopy has been developed for the analysis of postblast residues from 10 open-air explosions caused by 10 different explosives (TNT, RDX, PETN, TATP, HMTD, dynamite, black powder, ANFO, chloratite, and ammonal) commonly used in improvised explosive devices. The methodology for the determination of postblast particles from explosives consisted of examining the samples surfaces with both the naked eye, first, and microscopically (10× and 50×), immediately afterward; and finally, analyzing the selected residues by confocal Raman spectroscopy in order to identify the postblast particles from explosives. Interestingly, confocal Raman microscopy has demonstrated to be highly suitable to rapidly, selectively, and noninvasively analyze postblast microscopic particles from explosives up to the nanogram range. PMID:27281604

  2. MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES USING CONFOCAL LASER SCANNING MICROSCOPY

    EPA Science Inventory

    MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES USING CONFOCAL LASER SCANNING MICROSCOPY

    Robert M. Zucker Susan C. Jeffery and Sally D. Perreault

    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Prot...

  3. Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos

    EPA Science Inventory

    Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mous...

  4. Confocal microscopy studies of morphology and apoptosis: ovaries, limbs, embryos and insects

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ap...

  5. MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES, EMBRYOS AND FETAL LIMBS USING CONFOCAL MICROSCOPY

    EPA Science Inventory

    The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional section...

  6. Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  7. Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  8. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR QUANTIFYING CYTOMETRIC APPLICATIONS WITH SPECTROSCOPIC INSTRUMENTS

    EPA Science Inventory

    The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

  9. Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy.

    PubMed

    Babes, Liane; Kubes, Paul

    2016-01-01

    Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor-stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver. PMID:27581024

  10. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms

    PubMed Central

    Bouchard, Matthew B.; Voleti, Venkatakaushik; Mendes, César S.; Lacefield, Clay; Grueber, Wesley B.; Mann, Richard S.; Bruno, Randy M.; Hillman, Elizabeth M. C.

    2014-01-01

    We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae. PMID:25663846

  11. Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin

    NASA Astrophysics Data System (ADS)

    Song, Eunjoo; Ahn, YoonJoon; Ahn, Jinhyo; Ahn, Soyeon; Kim, Changhwan; Choi, Sanghoon; Boutilier, Richard Martin; Lee, Yongjoong; Kim, Pilhan; Lee, Ho

    2015-10-01

    We examined the optical clearing assisted confocal microscopy of the transgenic mouse skin. The pinna and dorsal skin were imaged with a confocal microscope after the application of glycerol and FocusClear. In case of the glycerol-treated pinna, the clearing was minimal due to the inefficient permeability. However, the imaging depth was improved when the pinna was treated with FocusClear. In case of dorsal skin, we were able to image deeply to the subcutaneous connective tissue with both agents. Various skin structures such as the vessel, epithelium cells, cartilage, dermal cells, and hair follicles were clearly imaged.

  12. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy.

    PubMed

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  13. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    PubMed Central

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  14. Sub-diffraction imaging with confocal fluorescence microscopy by stochastic photobleaching

    NASA Astrophysics Data System (ADS)

    Wang, Yifan; Kuang, Cuifang; Cai, Huanqing; Li, Shuai; Liu, Wei; Hao, Xiang; Ge, Jianhong; Liu, Xu

    2014-02-01

    We propose a single molecule localization method which takes advantage of stochastic photobleaching to improve the resolution of confocal fluorescence microscopy. By detecting the stochastic intensity loss of fluorophores, each fluorophore in the field can be localized. When all locations are known, a sub-diffraction image can be retrieved through single molecule localization algorithms. A confocal scheme is used to record the bleaching process of the sample. Each fluorophore can be localized from the recorded streaming followed by image subtraction. Compared with other single molecule localization concepts such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM), this method does not require a laser cycling equipment and the pixel size is no longer limited by the size of CCD. This technique works well with common fluorescent dyes and does not require the use of engineered photoactivatable proteins or photoswitchable synthetic dye pairs.

  15. Dual-detection confocal microscopy: high-speed surface profiling without depth scanning

    NASA Astrophysics Data System (ADS)

    Lee, Dong-Ryoung; Gweon, Dae-Gab; Yoo, Hongki

    2016-03-01

    We propose a new method for three-dimensional (3-D) imaging without depth scanning that we refer to as the dual-detection confocal microscopy (DDCM). Compared to conventional confocal microscopy, DDCM utilizes two pinholes of different sizes. DDCM generates two axial response curves which have different stiffness according to the pinhole diameters. The two axial response curves can draw the characteristics curve of the system which shows the relationship between the axial position of the sample and the intensity ratio. Utilizing the characteristic curve, the DDCM reconstructs a 3-D surface profile with a single 2-D scanning. The height of each pixel is calculated by the intensity ratio of the pixel and the intensity ratio curve. Since the height information can be obtained directly from the characteristic curve without depth scanning, a major advantage of DDCM over the conventional confocal microscopy is a speed. The 3-D surface profiling time is dramatically reduced. Furthermore, DDCM can measure 3-D images without the influence of the sample condition since the intensity ratio is independent of the quantum yield and reflectance. We present two types of DDCM, such as a fluorescence microscopy and a reflectance microscopy. In addition, we extend the measurement range axially by varying the pupil function. Here, we demonstrate the working principle of DDCM and the feasibility of the proposed methods.

  16. Cement paste surface roughness analysis using coherence scanning interferometry and confocal microscopy

    SciTech Connect

    Apedo, K.L.; Munzer, C.; He, H.; Montgomery, P.; Serres, N.; Fond, C.; Feugeas, F.

    2015-02-15

    Scanning electron microscopy and scanning probe microscopy have been used for several decades to better understand the microstructure of cementitious materials. Very limited work has been performed to date to study the roughness of cementitious materials by optical microscopy such as coherence scanning interferometry (CSI) and chromatic confocal sensing (CCS). The objective of this paper is to better understand how CSI can be used as a tool to analyze surface roughness and topography of cement pastes. Observations from a series of images acquired using this technique on both polished and unpolished samples are described. The results from CSI are compared with those from a STIL confocal microscopy technique (SCM). Comparison between both optical techniques demonstrates the ability of CSI to measure both polished and unpolished cement pastes. - Highlights: • Coherence scanning interferometry (CSI) was used to analyze cement paste surfaces. • The results from the CSI were compared with those from a confocal microscopy. • 3D roughness parameters were obtained using the window resizing method. • Polished and unpolished cement pastes were studied.

  17. CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

    PubMed Central

    Siegel, Nisan; Storrie, Brian; Bruce, Marc

    2016-01-01

    FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

  18. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

    PubMed Central

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-01-01

    We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

  19. Use of a white light supercontinuum laser for confocal interference-reflection microscopy

    PubMed Central

    Chiu, L-D; Su, L; Reichelt, S; Amos, WB

    2012-01-01

    Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460–700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser. PMID:22432542

  20. In vivo molecular and morphological imaging by real time confocal mini-microscopy

    NASA Astrophysics Data System (ADS)

    Goetz, Martin; Gregor, Sebastian; Fottner, Christian; Garcia-Lazaro, Jose; Schirrmacher, Esther; Kempski, Oliver; Bartenstein, Peter; Weber, Mathias; Biesterfeld, Stefan; Galle, Peter R.; Neurath, Markus F.; Kiesslich, Ralf

    2006-02-01

    We evaluated a newly developed miniaturized confocal laser microscopy probe for real-time in vivo molecular and morphological imaging of normal, inflammatory, and malignant tissue in rodents. In the rigid mini-microscopy probe (diameter 7 mm), a single line laser delivers an excitation wavelength of 488 nm. Optical slice thickness is 7 μm, lateral resolution 0.7 μm. The range of the z-axis is 0 - 250 μm below the tissue surface. Organ systems were examined in vivo in rodent models of human diseases. FITC-labeled Lycopersion esculentum lectin was injected or selected cell populations stained for molecular targeting. Morphological imaging was performed using fluorescein sodium, FITC-labeled dextran, and/or acriflavine hydrochloride. Cellular and subcellular details could be readily visualised in vivo at high resolution. Tissue characteristics of different organs were rendered at real time. Selective blood cell staining allowed observation of blood flow and cell migration. Inflammatory diseases such as hepatitis were diagnosed, and tumors were characterized under microscopic control in vivo. Confocal mini-microscopy allows real time in vivo molecular and morphological histologic imaging at high resolution of normal and diseased tissue. Since confocal microscopy is applicable to humans, this technology will have a high impact on different faculties in medicine.

  1. In-vivo immunofluorescence confocal microscopy of herpes simplex virus type 1 keratitis

    NASA Astrophysics Data System (ADS)

    Kaufman, Stephen C.; Laird, Jeffery A.; Beuerman, Roger W.

    1996-05-01

    The white-light confocal microscope offers an in vivo, cellular-level resolution view of the cornea. This instrument has proven to be a valuable research and diagnostic tool for the study of infectious keratitis. In this study, we investigate the direct visualization of herpes simplex virus type 1 (HSV-1)-infected corneal epithelium, with in vivo confocal microscopy, using HSV-1 immunofluorescent antibodies. New Zealand white rabbits were infected with McKrae strain of HSV-1 in one eye; the other eye of each rabbit was used as an uninfected control. Four days later, the rabbits were anesthetized and a cellulose sponge was applied to each cornea, and a drop of direct HSV fluorescein-tagged antibody was placed on each sponge every 3 to 5 minutes for 1 hour. Fluorescence confocal microscopy was then performed. The HSV-infected corneas showed broad regions of hyperfluorescent epithelial cells. The uninfected corneas revealed no background fluorescence. Thus, using the confocal microscope with a fluorescent cube, we were able to visualize HSV-infected corneal epithelial cells tagged with a direct fluorescent antibody. This process may prove to be a useful clinical tool for the in vivo diagnosis of HSV keratitis.

  2. Three-dimensional reconstruction of topological deformation in chiral nematic microspheres using fluorescence confocal polarizing microscopy.

    PubMed

    Guo, Jin-Kun; Song, Jang-Kun

    2016-04-01

    Chiral nematic droplets exhibit abundant topological defect structures, which have been intensively studied, both theoretically and experimentally. However, to observe and reconstruct the exact shape of three-dimensional (3D) defect structures has been a challenging task. In this study, we successfully reconstruct the 3D defect structures within a CLC microsphere with long helical pitches by combining polarized optical microscopy (POM) and laser scanning type fluorescence confocal polarizing microscopy (FCPM). The obtained confocal stack images provide us with the vertical location of disclination defects, to allow reconstruction of the full 3D structures. The reconstructed 3D structures can be viewed from different directions, providing a better understanding of the topological structure. Moreover, the defect lines are identified to be + 1 defects, different from the previous prediction. Thus, FCPM provides an excellent tool to study the complex topological configuration in microspheres, and fosters its potential applicability in new devices based on topologically structured soft media. PMID:27137028

  3. Three-dimensional resolution and contrast-enhanced confocal microscopy with array detection.

    PubMed

    Ge, Baoliang; Wang, Yifan; Huang, Yujia; Kuang, Cuifang; Fang, Yue; Xiu, Peng; Rong, Zihao; Liu, Xu

    2016-05-01

    What we believe is a novel method for improving confocal microscopy's resolution and contrast in 3D space is proposed. Based on a conventional confocal microscopy setup, we use an array detector composed of 32 photomultiplier tubes (PMTs) to replace one point-detector, where the location offset of each PMT caused a different effective point spread function (PSF). By applying array detection and the fluorescence emission difference method of an image with a solid PSF and another with a donut-shaped PSF, we can enhance lateral resolution about 27% in real time with only one scan, and improve the axial resolving ability by about 22% simultaneously. Experimental results of both fluorescent beads and living cells are presented to verify the applicability and effectiveness of our method. PMID:27128062

  4. Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

    PubMed

    Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

    2015-01-27

    Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy. PMID:25506739

  5. Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin

    PubMed Central

    Mortensen, Luke J.; Glazowski, Christopher E.; Zavislan, James M.; DeLouise, Lisa A.

    2011-01-01

    Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level. PMID:21698023

  6. Analysis of cell-tissue grafts under weightless conditions using confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Volova, L. T.; Milyakova, M. N.; Rossinskaya, V. V.; Boltovskaya, V. V.; Kulagina, L. N.; Kurganskaya, L. V.; Timchenko, P. E.; Timchenko, E. V.; Zherdeva Taskina, Larisa A.

    2015-03-01

    The research results of monitoring of viable cells in a cellular-tissue graft using confocal laser fluorescence microscopy at 488 nm and 561 nm with the use of fluorophore propidium iodide (propidium iodide, PI Sigma Aldrich USA) are presented. The processing of the received images was carried out using the software ANDOR. It is experimentally shown that the method of confocal fluorescence microscopy is one of the informational methods for detecting cells populated in a 3-D bio-carrier with a resolution of at least 400 nm. Analysis of the received micrographs suggests that the cells that were in a bio-carrier for 30 days in a synchronous ground-based experiment retained their viability compared to a similar space-based experiment in which the cells were hardly detected in a bio-carrier.

  7. Confocal Raman microscopy to monitor extracellular matrix during dental pulp stem cells differentiation

    NASA Astrophysics Data System (ADS)

    Salehi, Hamideh; Collart-Dutilleul, Pierre-Yves; Gergely, Csilla; Cuisinier, Frédéric J. G.

    2015-07-01

    Regenerative medicine brings promising applications for mesenchymal stem cells, such as dental pulp stem cells (DPSCs). Confocal Raman microscopy, a noninvasive technique, is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800 to 3000 cm-1 region (C-H stretching) and the 960 cm-1 peak (ν1 PO43-) were collected (to image cells and phosphate, respectively), and the ratio of two peaks 1660 over 1690 cm-1 (amide I bands) to measure the collagen cross-linking has been calculated. Raman spectra of DPSCs after 21 days differentiation reveal several phosphate peaks: ν1 (first stretching mode) at 960 cm-1, ν2 at 430 cm-1, and ν4 at 585 cm-1 and collagen cross-linking can also be calculated. Confocal Raman microscopy enables monitoring osteogenic differentiation in vitro and can be a credible tool for clinical stem cell based research.

  8. Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Collart-Dutilleul, P.-Y.; Gergely, C.; Cuisinier, F. J. G.

    2014-03-01

    Regenerative medicine brings a huge application for Mesenchymal stem cells such as Dental Pulp Stem Cells (DPSCs). Confocal Raman microscopy, a non-invasive, label free , real time and high spatial resolution imaging technique is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800-3000 cm-1 region (C-H stretching) and 960 cm-1 peak (phosphate PO4 3-) were collected. In Dental Pulp Stem Cells 21st day differentiated in buffer solution, phosphate peaks ν1 PO4 3- (first vibrational mode) at 960cm-1 and ν2 PO4 3- at 430cm-1 and ν4 PO4 3- at 585cm-1 are obviously present. Confocal Raman microscopy enables the detection of cell differentiation and it can be used to investigate clinical stem cell research.

  9. Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy

    NASA Astrophysics Data System (ADS)

    Sun, Hui; Kurtz, Ronald; Juhasz, Tibor

    2012-08-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial thickness intrascleral channels can be created with a femtosecond laser operating at a wavelength of 1700 nm. Such channels have the potential to increase outflow facility and reduce elevated IOP. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in human cadaver eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such channels. This demonstrates that concept of integrating femtosecond laser surgery, and two-photon and confocal imaging has the future potential for image-guided high-precision surgery in transparent and translucent tissue.

  10. Template confined synthesis of amorphous carbon nanotubes and its confocal Raman microscopy

    SciTech Connect

    Maity, Supratim; Roychowdhury, Tuhin; Chattopadhyay, Kalyan Kumar

    2014-04-24

    Amorphous carbon nanotubes (aCNTs) were synthesized by AAO (anodic aluminum oxide) template at a temperature 500 °C in nitrogen atmosphere using the citric acid as a carbon source without the help of any catalyst particles. Morphological analysis of the as prepared samples was carried out by field emission scanning electron microscopy (FESEM). Confocal Raman imaging has been studied and an attempt has been made to find out the graphitic (sp{sup 2}) and disordered phase of the CNTs.

  11. In vivo confocal microscopy in dermatology: from research to clinical application

    NASA Astrophysics Data System (ADS)

    Ulrich, Martina; Lange-Asschenfeldt, Susanne

    2013-06-01

    Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research.

  12. Single Fluorescent Molecule Confocal Microscopy: A New Tool for Molecular Biology Research and Biosensor Development

    SciTech Connect

    Darrow, C.; Huser, T.; Campos, C.; Yan, M.; Lane, S.; Balhorn, R.

    2000-03-09

    Our original proposal was presented to the LDRD committee on February 18, 1999. The revised proposal that followed incorporated changes that addressed the issues, concerns, and suggestions put forth by the committee members both during the presentation and in subsequent discussions we've had with individual committee members. The goal of the proposal was to establish an SMD confocal microscopy capability and technology base at LLNL. Here we report on our progress during the 6-month period for which funding was available.

  13. UNDERSTANDING THE EFFECTS OF SURFACTANT ADDITION ON RHEOLOGY USING LASER SCANNING CONFOCAL MICROSCOPY

    SciTech Connect

    White, T

    2007-05-08

    The effectiveness of three dispersants to modify rheology was examined using rheology measurements and laser scanning confocal microscopy (LSCM) in simulated waste solutions. All of the dispersants lowered the yield stress of the slurries below the baseline samples. The rheology curves were fitted reasonably to a Bingham Plastic model. The three-dimensional LSCM images of simulants showed distinct aggregates were greatly reduced after the addition of dispersants leading to a lowering of the yield stress of the simulated waste slurry solutions.

  14. Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy.

    PubMed

    Galli, Paolo; Strona, Giovanni; Villa, Anna Maria; Benzoni, Francesca; Fabrizio, Stefani; Doglia, Silvia Maria; Kritsky, Delane C

    2006-04-01

    A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy. PMID:16729702

  15. Application of confocal laser microscopy for monitoring mesh implants in herniology

    SciTech Connect

    Zakharov, V P; Belokonev, V I; Bratchenko, I A; Timchenko, P E; Vavilov, A V; Volova, L T

    2011-04-30

    The state of the surface of mesh implants and their encapsulation region in herniology is investigated by laser confocal microscopy. A correlation between the probability of developing relapses and the size and density of implant microdefects is experimentally shown. The applicability limits of differential reverse scattering for monitoring the post-operation state of implant and adjacent tissues are established based on model numerical experiments. (optical technologies in biophysics and medicine)

  16. In vivo confocal microscopy in dermatology: from research to clinical application.

    PubMed

    Ulrich, Martina; Lange-Asschenfeldt, Susanne

    2013-06-01

    Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research. PMID:23338938

  17. Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Han, Jason J.; Kunde, Yuliya A.; Hong-Geller, Elizabeth; Werner, James H.

    2014-01-01

    We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

  18. Analyzing cell structure and dynamics with confocal light scattering and absorption spectroscopic microscopy

    NASA Astrophysics Data System (ADS)

    Qiu, Le; Vitkin, Edward; Fang, Hui; Zaman, Munir M.; Andersson, Charlotte; Salahuddin, Saira; Modell, Mark D.; Freedman, Steven D.; Hanlon, Eugene B.; Itzkan, Irving; Perelman, Lev T.

    2007-02-01

    We recently developed a new microscopic optical technique capable of noninvasive analysis of cell structure and cell dynamics on the submicron scale [1]. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light scattering spectroscopy (LSS) and is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales on the order of 100 nm. To test the ability of CLASS microscopy to monitor cellular dynamics in vivo we performed experiments with human bronchial epithelial cells treated with DHA and undergoing apoptosis. The treated and untreated cells show not only clear differences in organelle spatial distribution but time sequencing experiments on a single cell show disappearance of certain types of organelles and change of the nuclear shape and density with the progression of apoptosis. In summary, CLASS microscopy provides an insight into metabolic processes within the cell and opens doors for the noninvasive real-time assessment of cellular dynamics. Noninvasive monitoring of cellular dynamics with CLASS microscopy can be used for a real-time dosimetry in a wide variety of medical and environmental applications that have no immediate observable outcome, such as photodynamic therapy, drug screening, and monitoring of toxins.

  19. Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy.

    PubMed

    Tan, Yuen-Fen; Leong, Chooi-Fun; Cheong, Soon-Keng

    2010-12-01

    Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs. PMID:21329180

  20. Combining confocal Raman microscopy and freeze-drying for quantification of substance penetration into human skin.

    PubMed

    Franzen, Lutz; Anderski, Juliane; Planz, Viktoria; Kostka, Karl-Heinz; Windbergs, Maike

    2014-12-01

    In the area of dermatological research, the knowledge of rate and extent of substance penetration into the human skin is essential not only for evaluation of therapeutics, but also for risk assessment of chemicals and cosmetic ingredients. Recently, confocal Raman microscopy emerged as a novel analytical technique for analysis of substance skin penetration. In contrast to destructive drug extraction and quantification, the technique is non-destructive and provides high spatial resolution in three dimensions. However, the generation of time-resolved concentration depth profiles is restrained by ongoing diffusion of the penetrating substance during analysis. To prevent that, substance diffusion in excised human skin can instantly be stopped at defined time points by freeze-drying the sample. Thus, combining sample preparation by freeze-drying with drug quantification by confocal Raman microscopy yields a novel analytical platform for non-invasive and quantitative in vitro analysis of substance skin penetration. This work presents the first proof-of-concept study for non-invasive quantitative substance depth profiling in freeze-dried excised human stratum corneum by confocal Raman microscopy. PMID:25219950

  1. Visualization and quantification of dentin structure using confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Kimura, Yuichi; Wilder-Smith, Petra B.; Krasieva, Tatiana B.; Arrastia-Jitosho, Anna-Marie A.; Liaw, Lih-Huei L.; Matsumoto, Koukichi

    1997-07-01

    Dentin was visualized using a new fluorescence technique and confocal laser scanning microscopy. Thirty extracted human teeth showing no clinical signs of caries were investigated. All teeth were horizontally sectioned to approximately 200 micrometers thickness and sections were subjected to different pretreatment conditions as follows: vacuum only, ultrasonication only, sodium hypochlorite only, sodium hypochlorite and vacuum, sodium hypochlorite and ultrasonication, and a combination of sodium hypochlorite, vacuum, and ultrasonication. Some samples were left untreated to serve as control. Following pretreatment, rhodamine 123 fluorescent dye was used for staining at concentrations ranging from 10-3 to 10-7 M for 1 to 24 h at pH 6.0, 6.5, or 7.4. Optical staining occurred at pH 7.4 and concentrations >= 10-5 M over 3 h or longer. Surface images obtained using confocal laser scanning microscopy were similar to those observed by scanning electron microscopy without the need for sample- altering conventional scanning electron microscope preparation techniques. Subsurface imaging to a depth of approximately 60 micrometers was achieved using confocal laser microscope techniques. This fluorescence technique offers a useful new alternative for visualization and quantification of dentin.

  2. In vivo confocal microscopy for the oral cavity: Current state of the field and future potential.

    PubMed

    Maher, N G; Collgros, H; Uribe, P; Ch'ng, S; Rajadhyaksha, M; Guitera, P

    2016-03-01

    Confocal microscopy (CM) has been shown to correlate with oral mucosal histopathology in vivo. The purposes of this review are to summarize what we know so far about in vivo CM applications for oral mucosal pathologies, to highlight some current developments with CM devices relevant for oral applications, and to formulate where in vivo CM could hold further application for oral mucosal diagnosis and management. Ovid Medline® and/or Google® searches were performed using the terms 'microscopy, confocal', 'mouth neoplasms', 'mouth mucosa', 'leukoplakia, oral', 'oral lichen planus', 'gingiva', 'cheilitis', 'taste', 'inflammatory oral confocal', 'mucosal confocal' and 'confocal squamous cell oral'. In summary, inclusion criteria were in vivo use of any type of CM for the human oral mucosa and studies on normal or pathological oral mucosa. Experimental studies attempting to identify proteins of interest and microorganisms were excluded. In total 25 relevant articles were found, covering 8 main topics, including normal oral mucosal features (n=15), oral dysplasia or neoplasia (n=7), inflamed oral mucosa (n=3), taste impairment (n=3), oral autoimmune conditions (n=2), pigmented oral pathology/melanoma (n=1), delayed type hypersensitivity (n=1), and cheilitis glandularis (n=1). The evidence for using in vivo CM in these conditions is poor, as it is limited to mainly small descriptive studies. Current device developments for oral CM include improved probe design. The authors propose that future applications for in vivo oral CM may include burning mouth syndrome, intra-operative mapping for cancer surgery, and monitoring and targeted biopsies within field cancerization. PMID:26786962

  3. The Unique Pollen Morphology of Duparquetia (Leguminosae: Caesalpinioideae): Developmental Evidence of Aperture Orientation Using Confocal Microscopy

    PubMed Central

    BANKS, HANNAH; FEIST-BURKHART, SUSANNE; KLITGAARD, BENTE

    2006-01-01

    • Background and Aims The phylogenetic affinities of the aberrant monotypic genus Duparquetia (subfamily Caesalpinioideae) are at present unresolved. Preliminary results from molecular analyses suggest a basal, isolated position among legumes. A study of Duparquetia pollen was carried out to provide further morphological characters to contribute to multi-data set analyses. Understanding the development of Duparquetia pollen was necessary to clarify the orientation of the apertures. • Methods Pollen grains and developing microspores were examined using light microscopy, confocal microscopy and scanning electron microscopy. Evidence for the orientation of the apertures was provided by the examination of microspores within developing tetrads, using (a) confocal microscopy to locate the position of the ectoapertures, and (b) light microscopy and Alcian blue stain to locate the position of the endoapertures. • Key Results Confocal microscopy has been used for the first time to examine developing microspores in order to obtain information on ectoapertures that was unavailable using other techniques. Pollen in Duparquetia develops in tetrahedral tetrads as in other eudicots, with the apertures arranged in a modified pattern following Fischer's rule. Pollen grains are asymmetrical and have one equatorial-encircling ectoaperture with two equatorial endoapertures, a unique feature in Leguminosae, and in eudicots. • Conclusions The pollen morphology of Duparquetia is so unusual that it provides little information to help determine its closest relatives. However, it does fit with a pattern of greater pollen morphological diversity in the first-branching caesalpinioid legume groups than in the more derived clades. The latitudinal ectoaperture of Duparquetia is unique within the Fabales and eudicot clades, resembling more closely the monosulcate pollen found in monocots and basal angiosperms; however, developmental patterns are recognizably similar to those of all other

  4. Evaluation of the therapeutic results of actinic keratosis treated with topical 5% fluorouracil by reflectance confocal laser microscopy: preliminary study*

    PubMed Central

    Ishioka, Priscila; Maia, Marcus; Rodrigues, Sarita Bartholomei; Marta, Alessandra Cristina; Hirata, Sérgio Henrique

    2015-01-01

    Topical treatment for actinic keratosis with 5% fluorouracil has a recurrence rate of 54% in 12 months of follow-up. This study analyzed thirteen actinic keratoses on the upper limbs through confocal microscopy, at the time of clinical diagnosis and after 4 weeks of treatment with fluorouracil. After the treatment was established and evidence of clinical cure was achieved, in two of the nine actinic keratoses, confocal microscopy enabled visualization of focal areas of atypical honeycomb pattern in the epidermis indicating therapeutic failure. Preliminary data suggest the use of confocal microscopy as a tool for diagnosis and therapeutic control of actinic keratosis. PMID:26131881

  5. Fast Imaging with Inelastically Scattered Electrons by Off-Axis Chromatic Confocal Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne

    2014-04-01

    We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840 eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

  6. Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

    NASA Astrophysics Data System (ADS)

    Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

    2006-02-01

    Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

  7. In vivo reflectance confocal microscopy of shave biopsy wounds: feasibility of intra-operative mapping of cancer margins

    PubMed Central

    Scope, A; Mahmood, U; Gareau, DS; Kenkre, M; Lieb, JA; Nehal, KS; Rajadhyaksha, M

    2010-01-01

    Background Reflectance confocal microscopy (RCM) images skin at cellular resolution and has shown utility for the diagnosis of nonmelanoma skin cancer in-vivo. Topical application of Aluminum Chloride (AlCl3) enhances contrast in RCM images by brightening nuclei. Objective To investigate feasibility of RCM imaging of shave biopsy wounds using AlCl3 as a contrast agent. Methods AlCl3 staining was optimized, in terms of concentration versus immersion time, on excised tissue ex-vivo. RCM imaging protocol was tested in patients undergoing shave biopsies. The RCM images were retrospectively analyzed and compared to the corresponding histopathology. Results For 35% AlCl3, routinely used for hemostasis in clinic, minimum immersion time was determined to be 1 minute. We identified 3 consistent patterns of margins on RCM mosaic images by varying depths: epidermal margins, peripheral dermal margins, and deep dermal margins. Tumour islands of basal cell carcinoma were identified at peripheral or deep dermal margins, correlating on histopathology with aggregates of neoplastic basaloid cells. Atypical cobblestone or honeycomb pattern were identified at the epidermal margins, correlating with a proliferation of atypical keratinocytes extending to biopsy margins. Conclusions RCM imaging of shave biopsy wounds is feasible and demonstrates the future possibility of intra-operative mapping in surgical wounds. PMID:20874785

  8. Influence of confocal scanning laser microscopy specific acquisition parameters on the detection and matching of speeded-up robust features.

    PubMed

    Stanciu, Stefan G; Hristu, Radu; Stanciu, George A

    2011-04-01

    The robustness and distinctiveness of local features to various object or scene deformations and to modifications of the acquisition parameters play key roles in the design of many computer vision applications. In this paper we present the results of our experiments on the behavior of a recently developed technique for local feature detection and description, Speeded-Up Robust Features (SURF), regarding image modifications specific to Confocal Scanning Laser Microscopy (CSLM). We analyze the repeatability of detected SURF keypoints and the precision-recall of their matching under modifications of three important CSLM parameters: pinhole aperture, photomultiplier (PMT) gain and laser beam power. During any investigation by CSLM these three parameters have to be modified, individually or together, in order to optimize the contrast and the Signal Noise Ratio (SNR), being also inherently modified when changing the microscope objective. Our experiments show that an important amount of SURF features can be detected at the same physical locations in images collected at different values of the pinhole aperture, PMT gain and laser beam power, and further on can be successfully matched based on their descriptors. In the final part, we exemplify the potential of SURF in CSLM imaging by presenting a SURF-based computer vision application that deals with the mosaicing of images collected by this technique. PMID:21349249

  9. Towards real-time image deconvolution: application to confocal and STED microscopy

    PubMed Central

    Zanella, R.; Zanghirati, G.; Cavicchioli, R.; Zanni, L.; Boccacci, P.; Bertero, M.; Vicidomini, G.

    2013-01-01

    Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings. PMID:23982127

  10. Taylor series expansion based multidimensional image reconstruction for confocal and 4pi microscopy

    NASA Astrophysics Data System (ADS)

    Dilipkumar, Shilpa; Pratim Mondal, Partha

    2013-08-01

    We propose and experimentally demonstrate a three-dimensional (3D) image reconstruction methodology based on Taylor series approximation (TSA) in a Bayesian image reconstruction formulation. TSA incorporates the requirement of analyticity in the image domain, and acts as a finite impulse response filter. This technique is validated on images obtained from widefield, confocal laser scanning fluorescence microscopy and two-photon excited 4pi (2PE-4pi) fluorescence microscopy. Studies on simulated 3D objects, mitochondria-tagged yeast cells (labeled with Mitotracker Orange) and mitochondrial networks (tagged with Green fluorescent protein) show a signal-to-background improvement of 40% and resolution enhancement from 360 to 240 nm. This technique can easily be extended to other imaging modalities (single plane illumination microscopy (SPIM), individual molecule localization SPIM, stimulated emission depletion microscopy and its variants).

  11. In Vivo Confocal Microscopy and Anterior Segment Optic Coherence Tomography Findings in Ocular Ochronosis

    PubMed Central

    Demirkilinc Biler, Elif; Guven Yilmaz, Suzan; Palamar, Melis; Hamrah, Pedram

    2015-01-01

    Purpose. To report clinical and in vivo confocal microscopy (IVCM) findings of two patients with ocular ochronosis secondary due to alkaptonuria. Materials and Methods. Complete ophthalmologic examinations, including IVCM (HRT II/Rostock Cornea Module, Heidelberg, Germany), anterior segment optical coherence tomography (AS-OCT) (Topcon 3D spectral-domain OCT 2000, Topcon Medical Systems, Paramus, NJ, USA), corneal topography (Pentacam, OCULUS Optikgeräte GmbH, Wetzlar, Germany), and anterior segment photography, were performed. Results. Biomicroscopic examination showed bilateral darkly pigmented lesions of the nasal and temporal conjunctiva and episclera in both patients. In vivo confocal microscopy of the lesions revealed prominent degenerative changes, including vacuoles and fragmentation of collagen fibers in the affected conjunctival lamina propria and episclera. Hyperreflective pigment granules in different shapes were demonstrated in the substantia propria beneath the basement membrane. AS-OCT of Case 1 demonstrated hyporeflective areas. Fundus examination was within normal limits in both patients, except tilted optic discs with peripapillary atrophy in one of the patients. Corneal topography, thickness, and macular OCT were normal bilaterally in both cases. Conclusion. The degenerative and anatomic changes due to ochronotic pigment deposition in alkaptonuria can be demonstrated in detail with IVCM and AS-OCT. Confocal microscopic analysis in ocular ochronosis may serve as a useful adjunct in diagnosis and monitoring of the disease progression. PMID:26788390

  12. Confocal reflectance quantitative phase microscopy system for cell biology studies (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Singh, Vijay Raj; So, Peter T. C.

    2016-03-01

    Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.

  13. Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo.

    PubMed Central

    Delaney, P M; King, R G; Lambert, J R; Harris, M R

    1994-01-01

    Fibre optic confocal imaging (FOCI) is a new type of microscopy which has been recently developed (Delaney et al. 1993). In contrast to conventional light microscopy, FOCI and other confocal techniques allow clear imaging of subsurface structures within translucent objects. However, unlike conventional confocal microscopes which are bulky (because of a need for accurate alignment of large components) FOCI allows the imaging end to be miniaturised and relatively mobile. FOCI is thus particularly suited for clear subsurface imaging of structures within living animals or subjects. The aim of the present study was to assess the suitability of using FOCI for imaging of subsurface structures within the colon, both in vitro (human and rat biopsies) and in vivo (in rats). Images were obtained in fluorescence mode (excitation 488 nm, detection above 515 nm) following topical application of fluorescein. By this technique the glandular structure of the colon was imaged. FOCI is thus suitable for subsurface imaging of the colon in vivo. Images Fig. 2 Fig. 3 PMID:8157487

  14. Confocal Raman microscopy for in depth analysis in the field of cultural heritage

    NASA Astrophysics Data System (ADS)

    Lorenzetti, G.; Striova, J.; Zoppi, A.; Castellucci, E. M.

    2011-05-01

    In the field of cultural heritage, the main concern when a sample is analyzed is its safeguard, and this means that non-destructive techniques are required. In this work, we show how confocal Raman microscopy (CRM) may be successfully applied in the study of works of art as a valuable alternative to other well established techniques. CRM with a metallurgical objective was tested for the in depth study of thin samples that are of interest in the field of cultural heritage. The sensitivity of the instrumentation was first evaluated by analyzing single layers of pure polyethylene terephthalate (PET) films having a thickness of 12, 25, and 50 μm, respectively, and a multilayer sample of polypropylene (PP) and polyethylene (PE). Subsequently, the technique was applied to the analysis of historical dyed cotton yarns in order to check whether it was possible to achieve a better discrimination of the fibres' signals for an easier identification. A substantial improvement of the signal to noise ratio was found in the confocal arrangement with respect to the non-confocal one, suggesting the use of this technique for this kind of analysis in the field of cultural heritage. Furthermore, Raman spectroscopy in confocal configuration was exploited in the evaluation of cleaning performed on the mural painting specimens, treated with acrylic resin (Paraloid B72). Confocal Raman experiments were performed before and after laser cleaning (at different conditions) in order to monitor the presence and to approximate the polymer thickness: the method proved to be a valid comparative tool in assessment of cleaning efficiencies.

  15. Design and analysis of multi-color confocal microscopy with a wavelength scanning detector.

    PubMed

    Do, Dukho; Chun, Wanhee; Gweon, Dae-Gab

    2012-05-01

    Spectral (or multi-color) microscopy has the ability to detect the fluorescent light of biological specimens with a broad range of wavelengths. Currently, the acousto-optic tunable filter (AOTF) is widely used in spectral microscopy as a substitute for a multiple-dichroic mirror to divide excitation and emission signals while maintaining sufficient light efficiency. In addition, systems which utilize an AOTF have a very fast switching speed and high resolution for wavelength selection. In this paper, confocal-spectral microscopy is proposed with a particular spectrometer design with a wavelength-scanning galvano-mirror. This enables the detection of broadband (480-700 nm) fluorescence signals by a single point detector (photomultiplier tube) instead of a CCD pixel array. For this purpose, a number of optical elements were applicably designed. A prism is used to amplify the dispersion angle, and the design of the relay optics matches the signals to the diameter of the wavelength-scanning galvano-mirror. Also, a birefringent material known as calcite is used to offset the displacement error at the image plane depending on the polarization states. The proposed multi-color confocal microscopy with the unique detection body has many advantages in comparison with commercial devices. In terms of the detection method, it can be easily applied to other imaging modalities. PMID:22667622

  16. Investigation of phosphatidylcholine enhancing FITC-insulin across buccal mucosa by confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Tian, Weiqun; Su, Li; Zeng, Shaoqun; Luo, Qingming; Gao, Qiuhua; Xu, Huibi

    2002-04-01

    The aim was to characterize the transport of fluorescein isothiocyanate (FITC)-labeled dextran and insulin with different resoluble compounds for peptides and proteins through buccal mucosa. The penetration rate of insulin molecules through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the rabbit buccal mucosa epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The confocal images of the distribution pattern of FITC-dextran and FITC-insulin showed that the paracellular route is the major pathway of FITC-dextran through buccal mucosa epithelium, the intra-cellular route is the major pathway of FITC-insulin through buccal mucosa epithelium. The permeation rate can be increased by co-administration of soybean phosphatidylcholine (SPC).

  17. Determination of sex by exfoliative cytology using acridine orange confocal microscopy: A short study

    PubMed Central

    Reddy, D Shyam Prasad; Sherlin, Herald J; Ramani, Pratibha; Prakash, P Ajay

    2012-01-01

    Context: Establishing individuality is an imperative aspect in any investigation procedure. Sometimes, in identifying an individual, it becomes necessary to determine the sex of that particular individual. Combining rapidity with reliability, an innovative idea has been put forward using a confocal microscope in exfoliative cytology. In the present study, we have determined the sex of the individual from buccal mucosal scrapings. The exfoliative cells were observed for Barr bodies under a confocal microscope, and the percentage of Barr-body-positive cells was determined. Aims: The main objective of this study is to assess confocal microscopy for the determination of sex by observing Barr bodies in the exfoliative cells of both men and women. Settings and Design: Samples of buccal mucosa smears were made followed by acridine orange staining. The stained slides were observed under a confocal microscope and the data obtained was subjected for statistical analysis, especially for mean and standard deviation. Materials and Methods: Samples of buccal mucosa smears from 20 men and 20 women were obtained by scraping with flat wooden sticks (exfoliative cytology). The smears were fixed in 100% alcohol for 15 min, followed by acridine orange (AO) staining as described by Von Bertalanffy et al. Smears stained with AO were examined under a confocal microscope and the percentage of Barr-body-positive cells was determined. Statistical Analysis Used: Data obtained was subjected for statistical analysis, especially for mean and standard deviation. Results: Two non-overlapping ranges for the percentage of Barr-body-positive cells have been obtained for men and women. It was observed that in the male samples, the percentage of Barr-body-positive cells ranged from 0-3%. In the female samples, the percentage of Barr-body-positive cells ranged from 18-72%, and all the females showed the presence of Barr bodies. Conclusion: The study showed that the presence of Barr body in buccal

  18. Advances in combined endoscopic fluorescence confocal microscopy and optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Risi, Matthew D.

    Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100 μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (˜1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography

  19. Scanning a microhabitat: plant-microbe interactions revealed by confocal laser microscopy

    PubMed Central

    Cardinale, Massimiliano

    2014-01-01

    No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology. PMID:24639675

  20. Probing the compressibility of tumor cell nuclei by combined atomic force-confocal microscopy

    NASA Astrophysics Data System (ADS)

    Krause, Marina; te Riet, Joost; Wolf, Katarina

    2013-12-01

    The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m-1, force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

  1. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rich, Thomas C.; Annamdevula, Naga; Britain, Andrea L.; Mayes, Samuel; Favreau, Peter F.; Leavesley, Silas J.

    2016-03-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors -- Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization -- whether epifluorescence or confocal microscopy -- may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

  2. Ti-6Al-4V electron beam weld qualification using laser scanning confocal microscopy

    SciTech Connect

    Wanjara, P. . E-mail: priti.wanjara@cnrc-nrc.gc.ca; Brochu, M.; Jahazi, M.

    2005-03-15

    Processing conditions for manufacturing Ti-6Al-4V components by welding using an electron beam source are known to influence the transformation microstructure in the narrow fusion and heat-affected zones of the weld region. This work examined the effect of multiple-sequence welding on the characteristics of the transformed beta microstructure, using laser scanning confocal microscopy to resolve the Widmanstaetten alpha-beta structure in the fusion zone. The evolution in the alpha interlamellar spacing and plate thickness with processing was then related to microhardness measurements in the weld region.

  3. Imaging Single ZnO Vertical Nanowire Laser Cavities using UV-Laser Scanning Confocal Microscopy

    SciTech Connect

    Gargas, D.J.; Toimil-Molares, M.E.; Yang, P.

    2008-11-17

    We report the fabrication and optical characterization of individual ZnO vertical nanowire laser cavities. Dilute nanowire arrays with interwire spacing>10 ?m were produced by a modified chemical vapor transport (CVT) method yielding an ideal platform for single nanowire imaging and spectroscopy. Lasing characteristics of a single vertical nanowire are presented, as well as high-resolution photoluminescence imaging by UV-laser scanning confocal microscopy. In addition, three-dimensional (3D) mapping of the photoluminescence emission performed in both planar and vertical dimensions demonstrates height-selective imaging useful for vertical nanowires and heteronanostructures emerging in the field of optoelectronics and nanophotonics.

  4. Spectrally encoded slit confocal microscopy using a wavelength-swept laser

    NASA Astrophysics Data System (ADS)

    Kim, Soocheol; Hwang, Jaehyun; Heo, Jung; Ryu, Suho; Lee, Donghak; Kim, Sang-Hoon; Oh, Seung Jae; Joo, Chulmin

    2015-03-01

    We present an implementation of spectrally encoded slit confocal microscopy. The method employs a rapid wavelength-swept laser as the light source and illuminates a specimen with a line focus that scans through the specimen as the wavelength sweeps. The reflected light from the specimen is imaged with a stationary line scan camera, in which the finite pixel height serves as a slit aperture. This scanner-free operation enables a simple and cost-effective implementation in a small form factor, while allowing for the three-dimensional imaging of biological samples.

  5. Confocal microscopy: A new tool for erosion measurements on large scale plasma facing components in tokamaks

    NASA Astrophysics Data System (ADS)

    Gauthier, E.; Brosset, C.; Roche, H.; Tsitrone, E.; Pégourié, B.; Martinez, A.; Languille, P.; Courtois, X.; Lallier, Y.; Salami, M.

    2013-07-01

    A diagnostic based on confocal microscopy was developed at CEA Cadarache in order to measure erosion on large plasma facing components during shutdown in situ in Tore Supra. This paper describes the diagnostic and presents results obtained on Beryllium and Carbon Fibre Composite (CFC) materials. Erosion in the range of 800 μm was found on one sector of the Toroidal Pumped Limiter (TPL) which provides, by integration to the full limiter a net carbon erosion of about 900 g over the period 2002-2007.

  6. Three-dimensional imaging of carbon nanostructures by scanning confocal electron microscopy

    NASA Astrophysics Data System (ADS)

    Hashimoto, Ayako; Shimojo, Masayuki; Mitsuishi, Kazutaka; Takeguchi, Masaki

    2009-10-01

    Although scanning confocal electron microscopy (SCEM) shows a promise for optical depth sectioning with high resolution, practical and theoretical problems have prevented its application to three-dimensional (3D) imaging. We employed a stage-scanning system in which only the specimen is moved three dimensionally under a fixed lens configuration, and an annular dark-field (ADF) aperture which blocks direct beams and selects only the scattered electrons. This ADF-SCEM improved depth resolution sufficiently to perform optical depth sectioning. Finally, we succeeded in demonstrating the 3D reconstruction of carbon nanocoils using ADF-SCEM.

  7. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Derely, L.; Vegh, A.-G.; Durand, J.-C.; Gergely, C.; Larroque, C.; Fauroux, M.-A.; Cuisinier, F. J. G.

    2013-03-01

    Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

  8. Insights into esophagus tissue architecture using two-photon confocal microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Nenrong; Wang, Yue; Feng, Shangyuan; Chen, Rong

    2013-08-01

    In this paper, microstructures of human esophageal mucosa were evaluated using the two-photon laser scanning confocal microscopy (TPLSCM), based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). The distribution of epithelial cells, muscle fibers of muscularis mucosae has been distinctly obtained. Furthermore, esophageal submucosa characteristics with cancer cells invading into were detected. The variation of collagen, elastin and cancer cells is very relevant to the pathology in esophagus, especially early esophageal cancer. Our experimental results indicate that the MPM technique has the much more advantages for label-free imaging, and has the potential application in vivo in the clinical diagnosis and monitoring of early esophageal cancer.

  9. Synchrotron radiation as a light source in confocal microscopy of biological processes

    NASA Astrophysics Data System (ADS)

    Gerritsen, Hans C.; van der Oord, C. J. R.; Levine, Yehudi K.; Munro, Ian H.; Myring, Wendy J.; Shaw, D. A.; Rommerts, Fokko F.

    1992-04-01

    A novel confocal microscope is presented using the Daresbury Synchrotron Radiation source as its light source. The broad spectrum of synchrotron radiation in combination with the UV compatible microscope allows the extension of confocal microscopy from the visible to the UV region down to about 200 nm. It is envisaged that structures separated by about 70 nm can be resolved at a wavelength of 200 nm. In addition, the tunability of synchrotron radiation affords the selective excitation of any specific fluorescent molecule at the maximum of the absorption band. This avoids the restriction of working at fixed laser lines. A further advantage of using synchrotron radiation is the realization of multiwavelength excitation. Test results using laser systems in the visible and in the UV are presented. Fluorescence images of test targets using UV excitation reveal the superior resolution of the microscope. Furthermore, images of Leydig cells incubated with a fluorescent cholesterol derivative whose maximum of absorption is at 325 nm are shown. These images cannot be produced by conventional confocal laser microscopes. Finally, promising preliminary results obtained with synchrotron radiation are presented.

  10. The application of dermal papillary rings in dermatology by in vivo confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Xiang, W. Z.; Xu, A. E.; Xu, J.; Bi, Z. G.; Shang, Y. B.; Ren, Q. S.

    2010-08-01

    Confocal laser scanning microscopy (CLSM) allows noninvasive visualization of human skin in vivo, without needing to fix or section the tissue. Melanocytes and pigmented keratinocytes at the level of the basal layer form bright dermal papillary rings which are readily amenable to identify in confocal images. Our purpose was to explore the role of dermal papillary rings in assessment of lesion location, the diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. Seventy-one patients were imaged with the VivaScope 1500 reflectance confocal microscope provided by Lucid, Inc. The results indicate that dermal papillary rings can assess the location of lesion; the application of dermal papillary rings can provide diagnostic support and differential diagnosis for vitiligo, nevus depigmentosus, tinea versicolor, halo nevus, common nevi, and assess the therapeutic efficacy of NBUVB phototherapy plus topical 0.1 percent tacrolimus ointment for vitiligo. In conclusion, our findings indicate that the dermal papillary rings play an important role in the assessment the location of lesion, diagnosis, differential diagnosis of lesions and assessment of therapeutic efficacy by in vivo CLSM. CLSM may be a promising tool for noninvasive examination in dermatology. However, larger studies are needed to expand the application of dermal papillary rings in dermatology.

  11. Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

    PubMed Central

    Tilli, Maddalena T; Parrish, Angela R; Cotarla, Ion; Jones, Laundette P; Johnson, Michael D; Furth, Priscilla A

    2008-01-01

    Background Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. Methods We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. Results In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. Conclusion In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary. PMID:18215290

  12. High numerical aperture injection-molded miniature objective for fiber-optic confocal reflectance microscopy

    NASA Astrophysics Data System (ADS)

    Chidley, Matthew Douglas

    This dissertation presents the design of a miniature injection-molded objective lens for a fiber-optic confocal reflectance microscope. This is part of an effort to demonstrate the ability to fabricate low cost, high performance biomedical optics for high resolution in vivo imaging. Disposable endoscopic microscope objectives could help in vivo confocal microscopy technology mature to enable large-scale clinical screening and detection of early cancers and pre-cancerous lesions. This five lens plastic objective has been tested as a stand-alone optical system and has been coupled to a confocal microscope for in vivo imaging of cells and tissue. Changing the spacing and rotation of the individual optical elements can compensate for fabrication inaccuracies and improve performance. An optical-bench testing system was constructed to allow interactive alignment during testing. The modulation transfer function (MTF) of the miniature objective lens is determined using the slanted-edge method. A custom MATLAB program, edgeMTF, was written to collect, analyze, and record test data. An estimated Strehl ratio of 0.64 and an MTF value of 0.70, at the fiber-optic bundle Nyquist frequency, have been obtained. The main performance limitations of the miniature objective are mechanical alignment and flow-induced birefringence. Annealing and experimental injection molding runs were conducted in effort to reduce birefringence.

  13. Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment

    NASA Astrophysics Data System (ADS)

    Rinehart, Matthew T.; LaCroix, Jeffrey; Henderson, Marcus; Katz, David; Wax, Adam

    2011-03-01

    The effectiveness of microbicidal gels, topical products developed to prevent infection by sexually transmitted diseases including HIV/AIDS, is governed by extent of gel coverage, pharmacokinetics of active pharmaceutical ingredients (APIs), and integrity of vaginal epithelium. While biopsies provide localized information about drug delivery and tissue structure, in vivo measurements are preferable in providing objective data on API and gel coating distribution as well as tissue integrity. We are developing a system combining confocal fluorescence microscopy with optical coherence tomography (OCT) to simultaneously measure local concentrations and diffusion coefficients of APIs during transport from microbicidal gels into tissue, while assessing tissue integrity. The confocal module acquires 2-D images of fluorescent APIs multiple times per second allowing analysis of lateral diffusion kinetics. The custom Fourier domain OCT module has a maximum a-scan rate of 54 kHz and provides depth-resolved tissue integrity information coregistered with the confocal fluorescence measurements. The combined system is validated by imaging phantoms with a surrogate fluorophore. Time-resolved API concentration measured at fixed depths is analyzed for diffusion kinetics. This multimodal system will eventually be implemented in vivo for objective evaluation of microbicide product performance.

  14. In vivo Confocal Microscopy Report after Lasik with Sequential Accelerated Corneal Collagen Cross-Linking Treatment

    PubMed Central

    Mazzotta, Cosimo; Balestrazzi, Angelo; Traversi, Claudio; Caragiuli, Stefano; Caporossi, Aldo

    2014-01-01

    We report the first pilot qualitative confocal microscopic analysis of a laser in situ keratomileusis (Lasik) treatment combined with sequential high-fluence accelerated corneal collagen cross-linking, denominated Lasik XTra, by means of HRT II laser scanning in vivo confocal microscopy after a 6-month follow-up. After obtaining approval from the Siena University Hospital Institutional Review Board, a 33-year-old female patient underwent a Lasik XTra procedure in her left eye. Confocal analysis demonstrated induced slight corneal microstructural changes by the interaction between UV-A, riboflavin and corneal stromal collagen, beyond the interface to a depth of 160 µm, without adverse events at the interface and endothelial levels. This application may be considered a prophylactic biomechanical treatment, stiffening the intermediate corneal stroma to prevent corneal ectasia and stabilizing the clinical results of refractive surgery. According to our preliminary experiences, this combined approach may be useful in higher-risk Lasik patients for hyperopic treatments, high myopia and lower corneal thicknesses. PMID:24847258

  15. Confocal Microscopy of Unfixed Breast Needle Core Biopsies: A Comparison to Fixed and Stained Sections

    PubMed Central

    2009-01-01

    Background Needle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM) is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has the potential to reduce the time from excision to diagnosis. Following imaging, specimens can still be submitted for standard histopathological preparation. Methods Needle core breast specimens from 49 patients were imaged at the time of biopsy. These lesions had been characterized under the Breast Imaging Reporting And Data System (BI-RADS) as category 3, 4 or 5. The core biopsies were imaged with the CSLM before fixation. Samples were treated with 5% citric acid and glycerin USP to enhance nuclear visibility in the reflectance confocal images. Immediately following imaging, the specimens were fixed in buffered formalin and submitted for histological processing and pathological diagnosis. CSLM images were then compared to the standard histology. Results The pathologic diagnoses by standard histology were 7 invasive ductal carcinomas, 2 invasive lobular carcinomas, 3 ductal carcinomas in-situ (CIS), 21 fibrocystic changes/proliferative conditions, 9 fibroadenomas, and 5 other/benign; two were excluded due to imaging difficulties. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images and were comparable to standard histologic sections of the same tissue. Conclusion CSLM is a technique with the potential to screen needle core biopsy specimens in real-time. The confocal images contained sufficient information to identify stromal reactions such as fibrosis and cellular proliferations such as intra-ductal and infiltrating carcinoma, and

  16. Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.

    PubMed

    Martial, Franck P; Hartell, Nicholas A

    2012-01-01

    Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium

  17. Confocal Microscopy and Molecular-Specific Optical Contrast Agents for the Detection of Oral Neoplasia

    PubMed Central

    Carlson, Alicia L.; Gillenwater, Ann M.; Williams, Michelle D.; El-Naggar, Adel K.; Richards-Kortum, R. R.

    2009-01-01

    Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium. PMID:17877424

  18. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

    PubMed

    Huang, Chao; Sachse, Frank B; Hitchcock, Robert W; Kaza, Aditya K

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  19. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

    PubMed Central

    Huang, Chao; Sachse, Frank B.; Hitchcock, Robert W.; Kaza, Aditya K.

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2±0.3% and 98.0±0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2±0.3% and 94.0±2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  20. Spatial distribution of perylenequinones in lichens and extended quinones in quincyte using confocal fluorescence microscopy.

    PubMed

    Mathey, A; Lukins, P B

    2001-02-01

    The application of confocal fluorescence microscopy and microspectrofluorimetry to the characterization of the distribution of organic compounds in bulk lichens and mineral structures is demonstrated. Perylenequinones and extended quinones were chosen as both model compounds and as the naturally occurring fluorophores. These molecules occur, respectively, in corticolous microlichens and in a pink-colored mineral called quincyte. The structures of quincyte and of the lichens Cryptothelium rhodotitton and Graphis hematites are described, and the possibilities of energy dissipation and photoprotection mechanisms in these lichens are discussed. This study also illustrates how, for a wide range of specimens, naturally occurring quinone fluorophores in the specimen can be exploited directly to yield chemical and structural information without using fluorescent labelling. These intrinsic quinonoid compounds have molecular fluorescence yields and laser damage thresholds comparable or superior to common microscopy dyes, and can therefore be used to obtain high-contrast 3D fluorescence imaging without the complications introduced by dye labelling. PMID:10936454

  1. Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty

    PubMed Central

    Wang, Dai; Song, Peng; Wang, Shuting; Sun, Dapeng; Wang, Yuexin; Zhang, Yangyang

    2016-01-01

    Purpose. To evaluate the changes of keratocytes and dendritic cells in the central clear graft by laser scanning in vivo confocal microscopy after penetrating keratoplasty (PK). Methods. Thirty adult subjects receiving PK at Shandong Eye Institute and with clear grafts and no sign of immune rejection after surgery were recruited into this study, and 10 healthy adults were controls. The keratocytes and dendritic cells in the central graft were evaluated by laser scanning confocal microscopy, as well as epithelium cells, keratocytes, corneal endothelium cells, and corneal nerves (especially subepithelial plexus nerves). Results. Median density of subepithelial plexus nerves, keratocyte density in each layer of the stroma, and density of corneal endothelium cells were all lower in clear grafts than in controls. The dendritic cells of five (16.7%) patients were active in Bowman's membrane and stromal membrane of the graft after PK. Conclusions. Activated dendritic cells and Langerhans cells could be detected in some of the clear grafts, which indicated that the subclinical stress of immune reaction took part in the chronic injury of the clear graft after PK, even when there was no clinical rejection episode. PMID:27034940

  2. Combining microtomy and confocal laser scanning microscopy for structural analyses of plant-fungus associations.

    PubMed

    Rath, Magnus; Grolig, Franz; Haueisen, Janine; Imhof, Stephan

    2014-05-01

    The serious problem of extended tissue thickness in the analysis of plant-fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl™. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant-fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf-fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant-fungus symbioses. PMID:24249491

  3. Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M.; Koch, Edmund

    2012-07-01

    Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

  4. Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain.

    PubMed

    Silvestri, L; Bria, A; Sacconi, L; Iannello, G; Pavone, F S

    2012-08-27

    Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies. PMID:23037106

  5. Dye-enhanced multimodal confocal microscopy for noninvasive detection of skin cancers in mouse models

    NASA Astrophysics Data System (ADS)

    Park, Jesung; Mroz, Pawel; Hamblin, Michael R.; Yaroslavsky, Anna N.

    2010-03-01

    Skin cancer is the most common form of human cancer. Its early diagnosis and timely treatment is of paramount importance for dermatology and surgical oncology. In this study, we evaluate the use of reflectance and fluorescence confocal microscopy for detecting skin cancers in an in-vivo trial with B16F10 melanoma and SCCVII squamous cell carcinoma in mice. For the experiments, the mice are anesthetized, then the tumors are infiltrated with aqueous solution of methylene blue and imaged. Reflectance images are acquired at 658 nm. Fluorescence is excited at 658 nm and registered in the range between 690 and 710 nm. After imaging, the mice are sacrificed. The tumors are excised and processed for hematoxylin and eosin histopathology, which is compared to the optical images. The results of the study indicate that in-vivo reflectance images provide valuable information on vascularization of the tumor, whereas the fluorescence images mimic the structural features seen in histopathology. Simultaneous dye-enhanced reflectance and fluorescence confocal microscopy shows promise for the detection, demarcation, and noninvasive monitoring of skin cancer development.

  6. Depth characterization of photopolymerized films by confocal Raman microscopy using an immersion objective.

    PubMed

    Courtecuisse, François; Dietlin, Céline; Croutxé-Barghorn, Céline; Van der Ven, Leendert G J

    2011-10-01

    The depth characterization of photopolymer films by confocal Raman microscopy is often troublesome due to refraction effects. To minimize these effects, we used an oil immersion objective and a method was developed to avoid penetration of the oil without damaging the sample surface. Since the surface may be sticky if oxygen in the air inhibits the photopolymerization, a protective layer could not be put onto the film. Therefore, the method consists in using a thin polypropylene foil as substrate for the coating and placing the sample upside down under the objective. In this manner, the immersion oil could be deposited on top of the polypropylene. The advantage of this setup is that the oil, polypropylene substrate, and photopolymer film have close refractive indices. Basic calculations showed that the depth resolution is hardly affected in that configuration and double-bond conversion profiles could be plotted as a function of reliable nominal depth. The validity of the methodology was confirmed by experiments carried out with a dry metallurgical objective on the sample surface, face up, where refraction effects are still minor. In addition, infrared spectroscopy, which was used to follow the photopolymerization, corroborated the Raman conversion of the films over their thickness. The confocal Raman microscopy method can be applied to various photopolymerized systems to characterize their behavior towards oxygen inhibition and other heterogeneities in conversion arising from inner filter effects or interactions between additives for instance. PMID:21986072

  7. Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death.

    PubMed

    Knight, Martin M; Roberts, Susan R; Lee, David A; Bader, Dan L

    2003-04-01

    Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2- transients and oscillations. Decreasing the power of the laser light decreased the percent-age of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was approximately 90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+ oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells. PMID:12661552

  8. Confocal Raman microscopy and fluorescent in situ hybridization - A complementary approach for biofilm analysis.

    PubMed

    Kniggendorf, Ann-Kathrin; Nogueira, Regina; Kelb, Christian; Schadzek, Patrik; Meinhardt-Wollweber, Merve; Ngezahayo, Anaclet; Roth, Bernhard

    2016-10-01

    We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples. PMID:27423128

  9. High throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

    PubMed Central

    Dumitriu, Dani; Rodriguez, Alfredo; Morrison, John H.

    2012-01-01

    Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high resolution confocal microscopy, deconvolution and image analysis using NeuronStudio. Recent technical advancements include better preservation of tissue resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual Z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically for the identification of both optimal resolution as well as highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing 3 experimental groups with 8 subjects in each can take as little as one month if optimized for speed, or approximately 4 to 5 months if the highest resolution and morphometric detail is sought. PMID:21886104

  10. Determination of nitric oxide mediating intracellular Ca2+ release on neurons based on confocal microscopy imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Liqin; Wang, Yuhua; He, Yipeng; Zeng, Yixiu; Zhang, Yanding; Xie, Shusen

    2014-09-01

    The gas NO is a ubiquitous intercellular messenger that modulates a wide range of physiological and pathophysiological functions. But few studies were made to study the role of NO in the Ca2+ release in dorsal root ganglion (DRG) neurons by confocal microscopy. Thus the objective of this study was to assess if NO has a role in Ca2+ signaling in DRG neurons using confocal microscopy combined with special fluorescence probe Fluo-3/AM. A 100 μM concentration of the NO donors (Sodium Nitroprusside, Dihydrate, SNP) and NO synthase inhibitor (NG-Monomethyl-L-arginine, Monoacetate salt, L-NMMA) was used in the study. Results showed that the fluorescence intensity increased rapidly after injecting SNP, which indicated that SNP could enhance intracellular Ca2+ release. And the fluorescence intensity shrank gradually with time and kept at a low level for quite a long period after loading with L-NMMA which indicated that L-NMMA could block intracellular Ca2+ release. All these results demonstrated that NO was involved in the regulation of intracellular Ca2+ release in the DRG neurons.

  11. Adaptive and Background-Aware GAL4 Expression Enhancement of Co-registered Confocal Microscopy Images.

    PubMed

    Trapp, Martin; Schulze, Florian; Novikov, Alexey A; Tirian, Laszlo; J Dickson, Barry; Bühler, Katja

    2016-04-01

    GAL4 gene expression imaging using confocal microscopy is a common and powerful technique used to study the nervous system of a model organism such as Drosophila melanogaster. Recent research projects focused on high throughput screenings of thousands of different driver lines, resulting in large image databases. The amount of data generated makes manual assessment tedious or even impossible. The first and most important step in any automatic image processing and data extraction pipeline is to enhance areas with relevant signal. However, data acquired via high throughput imaging tends to be less then ideal for this task, often showing high amounts of background signal. Furthermore, neuronal structures and in particular thin and elongated projections with a weak staining signal are easily lost. In this paper we present a method for enhancing the relevant signal by utilizing a Hessian-based filter to augment thin and weak tube-like structures in the image. To get optimal results, we present a novel adaptive background-aware enhancement filter parametrized with the local background intensity, which is estimated based on a common background model. We also integrate recent research on adaptive image enhancement into our approach, allowing us to propose an effective solution for known problems present in confocal microscopy images. We provide an evaluation based on annotated image data and compare our results against current state-of-the-art algorithms. The results show that our algorithm clearly outperforms the existing solutions. PMID:26743993

  12. In vivo fluorescence confocal microscopy: indocyanine green enhances the contrast of epidermal and dermal structures

    NASA Astrophysics Data System (ADS)

    Skvara, Hans; Kittler, Harald; Schmid, Johannes A.; Plut, Ulrike; Jonak, Constanze

    2011-09-01

    In recent years, in vivo skin imaging devices have been successfully implemented in skin research as well as in clinical routine. Of particular importance is the use of reflectance confocal microscopy (RCM) and fluorescence confocal microscopy (FCM) that enable visualization of the tissue with a resolution comparable to histology. A newly developed commercially available multi-laser device in which both technologies are integrated now offers the possibility to directly compare RCM with FCM. The fluorophore indocyanine green (ICG) was intradermally injected into healthy forearm skin of 10 volunteers followed by in vivo imaging at various time points. In the epidermis, accurate assessment of cell morphology with FCM was supplemented by identification of pigmented cells and structures with RCM. In dermal layers, only with FCM connective tissue fibers were clearly contoured down to a depth of more than 100 μm. The fluorescent signal still provided a favorable image contrast 24 and 48 hours after injection. Subsequently, ICG was applied to different types of skin diseases (basal cell carcinoma, actinic keratosis, seborrhoeic keratosis, and psoriasis) in order to demonstrate the diagnostic benefit of FCM when directly compared with RCM. Our data suggest a great impact of FCM in combination with ICG on clinical and experimental dermatology in the future.

  13. Corneal confocal microscopy reveals trigeminal small sensory fiber neuropathy in amyotrophic lateral sclerosis

    PubMed Central

    Ferrari, Giulio; Grisan, Enrico; Scarpa, Fabio; Fazio, Raffaella; Comola, Mauro; Quattrini, Angelo; Comi, Giancarlo; Rama, Paolo; Riva, Nilo

    2014-01-01

    Although subclinical involvement of sensory neurons in amyotrophic lateral sclerosis (ALS) has been previously demonstrated, corneal small fiber sensory neuropathy has not been reported to-date. We examined a group of sporadic ALS patients with corneal confocal microscopy, a recently developed imaging technique allowing in vivo observation of corneal small sensory fibers. Corneal confocal microscopy (CCM) examination revealed a reduction of corneal small fiber sensory nerve number and branching in ALS patients. Quantitative analysis demonstrated an increase in tortuosity and reduction in length and fractal dimension of ALS patients’ corneal nerve fibers compared to age-matched controls. Moreover, bulbar function disability scores were significantly related to measures of corneal nerve fibers anatomical damage. Our study demonstrates for the first time a corneal small fiber sensory neuropathy in ALS patients. This finding further suggests a link between sporadic ALS and facial-onset sensory and motor neuronopathy (FOSMN) syndrome, a rare condition characterized by early sensory symptoms (with trigeminal nerve distribution), followed by wasting and weakness of bulbar and upper limb muscles. In addition, the finding supports a model of neurodegeneration in ALS as a focally advancing process. PMID:25360111

  14. Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source.

    PubMed

    Träutlein, D; Adler, F; Moutzouris, K; Jeromin, A; Leitenstorfer, A; Ferrando-May, E

    2008-03-01

    The performance of a confocal microscopy setup based on a single femtosecond fiber system is explored over a broad range of pump wavelengths for both linear and nonlinear imaging techniques. First, the benefits of a laser source in linear fluorescence excitation that is continuously tunable over most of the visible spectrum are demonstrated. The influences of subpicosecond pulse durations on the bleaching behavior of typical fluorophores are discussed. We then utilize the tunable near-infrared output of the femtosecond system in connection with a specially designed prism compressor for dispersion control. Pulses as short as 33 fs are measured in the confocal region. As a consequence, 2 mW of average power are sufficient for two-photon microscopy in an organotypic sample from the mouse brain. This result shows great prospect for deep-tissue imaging in the optimum transparency window around 1100 nm. In a third experiment, we prove that our compact setup is powerful enough to exploit even higher-order nonlinearities such as three-photon absorption that we use to induce spatially localized photodamage in DNA. PMID:19343635

  15. Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2012-12-01

    Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

  16. Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network.

    PubMed

    Alves da Rocha, Roney; Paiva, Igor Moura; Anjos, Virgílio; Furtado, Marco Antônio Moreira; Bell, Maria José Valenzuela

    2015-06-01

    In this work, we assessed the use of confocal Raman microscopy and artificial neural network as a practical method to assess and quantify adulteration of fluid milk by addition of whey. Milk samples with added whey (from 0 to 100%) were prepared, simulating different levels of fraudulent adulteration. All analyses were carried out by direct inspection at the light microscope after depositing drops from each sample on a microscope slide and drying them at room temperature. No pre- or posttreatment (e.g., sample preparation or spectral correction) was required in the analyses. Quantitative determination of adulteration was performed through a feed-forward artificial neural network (ANN). Different ANN configurations were evaluated based on their coefficient of determination (R2) and root mean square error values, which were criteria for selecting the best predictor model. In the selected model, we observed that data from both training and validation subsets presented R2>99.99%, indicating that the combination of confocal Raman microscopy and ANN is a rapid, simple, and efficient method to quantify milk adulteration by whey. Because sample preparation and postprocessing of spectra were not required, the method has potential applications in health surveillance and food quality monitoring. PMID:25828656

  17. Localization and movement of mineral oil in plants by fluorescence and confocal microscopy.

    PubMed

    Tan, B L; Sarafis, V; Beattie, G A C; White, R; Darley, E M; Spooner-Hart, R

    2005-10-01

    Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed. PMID:16118255

  18. Mosaicism

    MedlinePlus

    ... A diagnosis of mosaicism may cause confusion and uncertainty. A genetic counselor may help answer any questions ... member of Hi-Ethics and subscribes to the principles of the Health on the Net Foundation (www. ...

  19. Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

    PubMed Central

    Kumar, Deepak; Sahoo, Dipak K.; Maiti, Indu B.; Dey, Nrisingha

    2011-01-01

    Background Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. Methodology/Principal Findings We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. Conclusion and Significance We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety

  20. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart

    PubMed Central

    Huang, Chao; Kaza, Aditya K.; Hitchcock, Robert W.; Sachse, Frank B.

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery. PMID:25309455

  1. Correcting spherical aberrations in confocal light sheet microscopy: a theoretical study.

    PubMed

    Silvestri, L; Sacconi, L; Pavone, F S

    2014-07-01

    In the last years, fluorescence light sheet microscopy has attracted an increasing interest among the microscopy community. One of the most promising applications of this technique is the reconstruction of macroscopic biological specimens with microscopic resolution, without physical sectioning. To this aim, light sheet microscopy is combined with clearing protocols based on refractive index matching, which render the tissue transparent. However, these protocols lead to a huge drop in the fluorescence signal, limiting their practical applicability. The reduction of signal to background ratio is commonly ascribed to chemical degradation of the fluorophores by the organic solvents used for clearing. This view however completely neglects another important factor of contrast loss, i.e., optical aberrations. In fact, commercially available objectives suitable for light sheet microscopy are not designed for the refractive index of the clearing solutions, and this mismatch introduces severe spherical aberration. Here we simulated the aberrated point spread function (PSF) of a light sheet microscope with confocal slit detection. We investigated the variation of the PSF as a function of objective numerical aperture (NA) and of imaging depth inside the clearing solution. We also explored the possibility of correcting such spherical aberration by introducing extra optical devices in the detection path. By correcting up to the second order spherical aberration, a quasi-diffraction-limited regime can be recovered, and image quality is restored. PMID:24395714

  2. Confocal Raman microscopy for investigation of the level of differentiation in living neuroblastoma tumor cells

    NASA Astrophysics Data System (ADS)

    Scalfi-Happ, Claudia; Jauss, Andrea; Hollricher, Olaf; Fulda, Simone; Hauser, Carmen; Steiner, Rudolf; Rück, Angelika

    2007-07-01

    The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods. In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumor in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor neuroblastoma cell differentiation. Neuroblastoma cells at different degrees of differentiation were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation. Integration time per spectrum was 80-100 ms. A lateral resolution in submicrometer range was achieved by using a 60x water immersion lens with a numerical aperture of 1,0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands, by basis analysis using reference spectra or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images providing insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for differentiation. The results of this work may have applications in the

  3. Modeling of Fibrin Gels Based on Confocal Microscopy and Light-Scattering Data

    PubMed Central

    Magatti, Davide; Molteni, Matteo; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

    2013-01-01

    Fibrin gels are biological networks that play a fundamental role in blood coagulation and other patho/physiological processes, such as thrombosis and cancer. Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3–4. Although in the confocal images the hydrated fibers appear to be quite straight (mass fractal dimension Dm = 1), for the overall system 1confocal images, we developed a method to generate three-dimensional (3D) in silico gels made of cylindrical sticks of diameter d, density ρ, and average length 〈L〉, joined at randomly distributed nodal points. The resulting 3D network strikingly resembles real fibrin gels and can be sketched as an assembly of densely packed fractal blobs, i.e., regions of size ξ, where the fiber concentration is higher than average. The blobs are placed at a distance ξ0 between their centers of mass so that they are overlapped by a factor η = ξ/ξ0 and have Dm ∼1.2–1.6. The in silico gels’ structure is quantitatively analyzed by its 3D spatial correlation function g3D(r) and corresponding power spectrum I(q) = FFT3D[g3D(r)], from which ρ, d, Dm, η, and ξ0 can be extracted. In particular, ξ0 provides an excellent estimate of the gel mesh size. The in silico gels’ I(q) compares quite well with real gels’ elastic light-scattering measurements. We then derived an analytical form factor for accurately fitting the scattering data, which allowed us to directly recover the gels’ structural parameters. PMID:23473498

  4. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo.

    PubMed

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-02-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  5. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

    PubMed Central

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J.; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-01-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  6. Prototype study on a miniaturized dual-modality imaging system for photoacoustic microscopy and confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Sung-Liang; Xie, Zhixing; Guo, L. Jay; Wang, Xueding

    2014-03-01

    It is beneficial to study tumor angiogenesis and microenvironments by imaging the microvasculature and cells at the same time. Photoacoustic microscopy (PAM) is capable of sensitive three-dimensional mapping of microvasculature, while fluorescence microscopy may be applied to assessment of tissue pathology. In this work, a fiber-optic based PAM and confocal fluorescence microscopy (CFM) dual-modality imaging system was designed and built, serving as a prototype of a miniaturized dual-modality imaging probe for endoscopic applications. As for the design, we employed miniature components, including a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector. The system resolutions were calibrated as 8.8 μm in the lateral directions for both PAM and CFM, and 19 μm and 53 μm in the axial direction for PAM and CFM, respectively. Images of the animal bladders ex vivo were demonstrated to show the ability of the system in imaging not only microvasculature but also cellular structure.

  7. Fluorescence imaging and time-resolved spectroscopy of steroid using confocal synchrotron radiation microscopy

    NASA Astrophysics Data System (ADS)

    Gerritsen, Hans C.; van der Oord, C. J. R.; Levine, Yehudi K.; Munro, Ian H.; Jones, Gareth R.; Shaw, D. A.; Rommerts, Fokko F.

    1994-08-01

    The Confocal Synchrotron Radiation Microscope at Daresbury was used in a study of the transport and distribution of the steroid Coumestrol in single Leydig cells. The broad spectrum of synchrotron radiation in combination with UV compatible microscope optics affords the extension of confocal microscopy from the visible to the UV region down to about 200 nm. Consequently fluorescent molecules with absorption bands in the UV can be imaged. In addition the pulsed nature of the light source allows us to perform time-resolved fluorescence spectroscopy experiments on microscopic volumes. Coumestrol is a naturally fluorescing plant steroid exhibiting estrogenic activity. In physiological environments it has an absorption peak in the UV at 340 nm and it emits around 440 nm. First results indicate that the Coumestrol transport through the cell membrane is diffusion limited. The weak fluorescence observed in the nuclei of the Leydig cells may be due to fluorescence quenching arising from the interaction of the Coumesterol with nuclear components. However, micro-volume time-resolved fluorescence spectroscopy experiments on cell nuclei have revealed the same decay behavior for Coumesterol in both the cytoplasm and nucleus of the cells.

  8. Structure and chemical composition of the dentin-enamel junction analyzed by Confocal Raman Microscopy

    NASA Astrophysics Data System (ADS)

    Desoutter, A.; Salehi, H.; Slimani, A.; Marquet, P.; Jacquot, B.; Tassery, H.; Cuisinier, F. J. G.

    2014-02-01

    The structure and chemical composition of the human dentin-enamel junction (DEJ) was studied using confocal Raman microscopy - a chemical imaging technique. Slices of non-fixed, sound teeth were prepared with an Isomet diamond saw and scanned with Witec Alpha300R system. The combination of different characteristics peaks of phosphate, carbonate and organic matrix (respectively 960, 1072 and 1545 cm-1), generates images representing the chemical composition of the DEJ area. Images are also calculated using peak ratios enabling precise determination of the chemical composition across the DEJ. Then, with two characterized peaks, different pictures are calculated to show the ratio of two components. The images of the spatial distribution of mineral phosphate (960cm-1) to organic matrix (1545 cm-1) ratios, mineral carbonates (1072cm-1) to mineral phosphate ratios; and mineral carbonates to organic matrix ratios were reconstructed. Cross sectional and calculated graphic profile show the variations of the different chemical component ratios through the enamel and the dentin. Phosphate to organic ratio shows an accumulation of organic material under the enamel surface. The cross sectional profile of these pictures shows a high phosphate content compared to enamel in the vicinity of the DEJ. The Confocal Raman imaging technique can be used to further provide full chemical imaging of tooth, particularly of the whole DEJ and to study enamel and dentin decay.

  9. Fault localization and analysis in semiconductor devices with optical-feedback infrared confocal microscopy

    SciTech Connect

    Sarmiento, Raymund; Cemine, Vernon Julius; Tagaca, Imee Rose; Salvador, Arnel; Mar Blanca, Carlo; Saloma, Caesar

    2007-11-01

    We report on a cost-effective optical setup for characterizing light-emitting semiconductor devices with optical-feedback confocal infrared microscopy and optical beam-induced resistance change.We utilize the focused beam from an infrared laser diode to induce local thermal resistance changes across the surface of a biased integrated circuit (IC) sample. Variations in the multiple current paths are mapped by scanning the IC across the focused beam. The high-contrast current maps allow accurate differentiation of the functional and defective sites, or the isolation of the surface-emittingp-i-n devices in the IC. Optical beam-induced current (OBIC) is not generated since the incident beam energy is lower than the bandgap energy of the p-i-n device. Inhomogeneous current distributions in the IC become apparent without the strong OBIC background. They are located at a diffraction-limited resolution by referencing the current maps against the confocal reflectance image that is simultaneously acquired via optical-feedback detection. Our technique permits the accurate identification of metal and semiconductor sites as well as the classification of different metallic structures according to thickness, composition, or spatial inhomogeneity.

  10. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.