A Case of Bilateral Descemet's Membrane and Subepithelial Opacity: In vivo Laser Confocal Microscopic Study.

PubMed

Hatta, Yukiko; Yokogawa, Hideaki; Kobayashi, Akira; Torisaki, Makoto; Sugiyama, Kazuhisa

2013-01-01

To report the in vivo laser confocal microscopy findings from a patient with Descemet's membrane and subepithelial opacity OU. A healthy 41-year-old male with Descemet's membrane and subepithelial opacity OU was studied. Routine ophthalmic examination, standard slit-lamp biomicroscopy, and in vivo laser confocal microscopic analysis of the entire corneal layer were performed. Slit-lamp biomicroscopy revealed subepithelial opacity in the mid-peripheral to peripheral cornea and numerous opacities located at the level of Descemet's membrane. It was difficult to distinguish the precise histological location of the opacity. In vivo laser confocal microscopy showed numerous hyperreflective particles in the subepithelium to superficial stroma and hyperreflectivity of Descemet's membrane. No abnormalities could be detected in the epithelial cell layer, midstromal layer, deep stromal layer, or endothelial cell layer. Although the origin of the corneal opacities was unclear, in vivo laser confocal microscopy was useful for observing microstructural abnormalities in a case of Descemet's membrane and subepithelial opacity.

A Case of Bilateral Descemet's Membrane and Subepithelial Opacity: In vivo Laser Confocal Microscopic Study

PubMed Central

Hatta, Yukiko; Yokogawa, Hideaki; Kobayashi, Akira; Torisaki, Makoto; Sugiyama, Kazuhisa

2013-01-01

Purpose To report the in vivo laser confocal microscopy findings from a patient with Descemet's membrane and subepithelial opacity OU. Case Report A healthy 41-year-old male with Descemet's membrane and subepithelial opacity OU was studied. Routine ophthalmic examination, standard slit-lamp biomicroscopy, and in vivo laser confocal microscopic analysis of the entire corneal layer were performed. Slit-lamp biomicroscopy revealed subepithelial opacity in the mid-peripheral to peripheral cornea and numerous opacities located at the level of Descemet's membrane. It was difficult to distinguish the precise histological location of the opacity. In vivo laser confocal microscopy showed numerous hyperreflective particles in the subepithelium to superficial stroma and hyperreflectivity of Descemet's membrane. No abnormalities could be detected in the epithelial cell layer, midstromal layer, deep stromal layer, or endothelial cell layer. Conclusion Although the origin of the corneal opacities was unclear, in vivo laser confocal microscopy was useful for observing microstructural abnormalities in a case of Descemet's membrane and subepithelial opacity. PMID:23626574

Improved axial point spread function in a two-frequency laser scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Wu, Jheng-Syong; Chung, Yung-Chin; Chien, Jun-Jei; Chou, Chien

2018-01-01

A two-frequency laser scanning confocal fluorescence microscope (TF-LSCFM) based on intensity modulated fluorescence signal detection was proposed. The specimen-induced spherical aberration and scattering effect were suppressed intrinsically, and high image contrast was presented due to heterodyne interference. An improved axial point spread function in a TF-LSCFM compared with a conventional laser scanning confocal fluorescence microscope was demonstrated and discussed.

In vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta

PubMed Central

Kobayashi, Akira; Higashide, Tomomi; Yokogawa, Hideaki; Yamazaki, Natsuko; Masaki, Toshinori; Sugiyama, Kazuhisa

2014-01-01

Objective To report the in vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta (OI) with special attention to the abnormality of Bowman’s layer and sub-Bowman’s fibrous structures (K-structures). Patients and methods Two patients (67-year-old male and his 26-year-old son) with OI type I were included in this study. Slit lamp biomicroscopic and in vivo laser confocal microscopic examinations were performed for both patients. Central corneal thickness and central endothelial cell density were also measured. Results Although the corneas looked clear with normal endothelial density for both eyes in both patients, they were quite thin (386 μm oculus dexter (OD) (the right eye) and 384 μm oculus sinister (OS) (the left eye) in the father and 430 μm OD and 425 μm OS in the son). In both patients, slit lamp biomicroscopic and in vivo laser confocal microscopic examination showed similar results. Anterior corneal mosaics produced by rubbing the eyelid under fluorescein were completely absent in both eyes. In vivo laser confocal microscopy revealed an absent or atrophic Bowman’s layer; a trace of a presumed Bowman’s layer and/or basement membrane was barely visible with high intensity. Additionally, K-structures were completely absent in both eyes. Conclusion The absence of K-structures and fluorescein anterior corneal mosaics strongly suggested an abnormality of Bowman’s layer in these OI patients. PMID:24591812

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.

Confocal microscopy to guide laser ablation of basal cell carinoma: a preliminary feasibility study

NASA Astrophysics Data System (ADS)

Larson, Bjorg A.; Sierra, Heidy; Chen, Jason; Rajadhyaksha, Milind

2013-03-01

Laser ablation may be a promising method for removal of skin lesions, with the potential for better cosmetic outcomes and reduced scarring and infection. An obstacle to implementing laser ablation is that the treatment leaves no tissue for histopathological analysis. Pre-operative and intra-operative mapping of BCCs using confocal microscopy may guide the ablation of the tumor until all tumor is removed. We demonstrate preliminary feasibility of confocal microscopy to guide laser ablation of BCCs in freshly excised tissue from Mohs surgery. A 2940 nm Er:YAG laser provides efficient ablation of tumor with reduced thermal damage to the surrounding tissue.

In vivo laser confocal microscopic analysis of murine cornea and lens microstructures.

PubMed

Yuasa, Masashi; Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2008-01-01

The purpose of the current study is to investigate in vivo microstructures of anterior segments of normal murine eyes by new-generation in vivo laser confocal microscopy. Twenty-six corneas and lenses from 13 mice were analyzed by in vivo laser confocal microscopy. Murine corneal superficial cells formed a polygonal cell pattern, with a mean cell density of 577 +/- 115 cells/mm2 (mean +/- standard deviation). Corneal basal epithelial cells had dark cytoplasm and were closely organized (9,312 +/- 1,777 cells/mm2). Sub-basal nerve fiber bundles were arranged in a whorl pattern, with both clockwise and counter-clockwise patterns. In the stroma, keratocytes were observed as numerous reflective stellate structures. The endothelial cells were organized in a honeycomb pattern (2,463 +/- 292 cells/mm2). Deeper inside the eye, murine lens epithelial cells were organized in a regular pattern (4,168 +/- 636 cells/mm2) and numerous lens fibers were observed. In vivo laser confocal microscopy can provide high-resolution images of all corneal layers and lens structures of mice without sacrificing animals or tissue preparation.

A Novel Method of Diagnosing Aberrant Pancreas: Needle-based Confocal Laser Endomicroscopy.

PubMed

Yasuda, Muneji; Hara, Kazuo; Kurita, Yusuke; Tanaka, Hiroki; Obata, Masahiro; Kuraoka, Naosuke; Matsumoto, Shimpei; Ito, Ayako; Iwaya, Hiromichi; Toriyama, Kazuhiro; Okuno, Nozomi; Kuwahara, Takamichi; Hijioka, Susumu; Mizuno, Nobumasa; Onishi, Sachiyo; Hirayama, Yutaka; Ishihara, Makoto; Tanaka, Tsutomu; Tajika, Masahiro; Niwa, Yasumasa

2018-05-18

Aberrant pancreas is defined as pancreatic tissue present outside of the pancreas and is often found incidentally during esophagogastroduodenoscopy. Obtaining sufficient tissue to differentiate aberrant pancreas from other subepithelial lesions is sometimes difficult. Due to the lack of a definitive diagnosis, patients often undergo unnecessary surgery. We herein report the first case of aberrant pancreas in which the concomitant use of needle-based probe confocal laser endomicroscopy and fine-needle aspiration supported the final diagnosis. Needle-based probe confocal laser endomicroscopy provides a real-time in vivo histopathology evaluation and may be a feasible means of diagnosing aberrant pancreas.

Confocal Fabry-Perot interferometer for frequency stabilization of laser

NASA Astrophysics Data System (ADS)

Pan, H.-J.; Ruan, P.; Wang, H.-W.; Li, F.

2011-02-01

The frequency shift of laser source of Doppler lidar is required in the range of a few megahertzs. To satisfy this demand, a confocal Fabry-Perot (F-P) interferometer was manufactured as the frequency standard for frequency stabilization. After analyzing and contrasting the center frequency shift of confocal Fabry-Perot interferometers that are made of three different types of material with the change of temperature, the zerodur material was selected to fabricate the interferometer, and the cavity mirrors were optically contacted onto the end of spacer. The confocal Fabry-Perot interferometer was situated within a double-walled chamber, and the change of temperature in the chamber was less than 0.01 K. The experimental results indicate that the free spectral range is 500 MHz, the full-width at half maximum is 3.33 MHz, and the finesse is 150.

CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE OVARIES

EPA Science Inventory

Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse Ovaries. Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle...

In vivo laser confocal microscopy after non-Descemet's stripping automated endothelial keratoplasty.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2009-07-01

To investigate in vivo corneal changes in patients with bullous keratopathy who underwent non-Descemet's stripping automated endothelial keratoplasty (nDSAEK) with the use of laser confocal microscopy. Single-center, prospective clinical study. Ten eyes (10 patients; 3 men and 7 women; mean age, 73.5+/-6.6 years [mean+/-standard deviation]) with bullous keratopathy were evaluated in this study. In vivo laser confocal microscopy was performed before and 1, 3, and 6 months after nDSAEK. Selected confocal images of corneal layers were evaluated qualitatively and quantitatively for degree of haze and density of deposits. Before surgery, the following were observed in all patients: corneal epithelial edema, subepithelial haze, keratocytes in a honeycomb pattern, and tiny needle-shaped materials in the stroma. After nDSAEK, subepithelial haze, donor-recipient interface haze, and interface particles were observed in all measurable cases; postoperative haze, interface particles, and needle-shaped materials decreased statistically significantly (P<0.05) over the course of follow-up. In addition, hyperreflective giant interface particles were observed after nDSAEK in all patients. In vivo laser confocal microscopy can identify subclinical corneal abnormalities after nDSAEK such as subepithelial haze, host-recipient interface haze, host stromal needle-shaped materials, and host-recipient interface particles with characteristic giant particles. Further studies with this technology in a large number of patients and long-term follow-up are needed to understand fully the long-term corneal stromal changes after nDSAEK.

Combined use of confocal laser scanning microscopy (CLSM) and Raman microscopy (RM): investigations on EPS-Matrix.

PubMed

Wagner, Michael; Ivleva, Natalia P; Haisch, Christoph; Niessner, Reinhard; Horn, Harald

2009-01-01

Confocal laser scanning microscopy (CLSM) was applied in combination with Raman microscopy (RM) for the characterization of heterotrophic biofilms. Compared to CLSM, RM allows for a deeper insight into the chemical structure of extracellular polymeric substances (EPS) of the biofilm matrix. A low load of glucose (2 g m(-2)d(-1)) was applied as substrate to ensure small growth rates of the heterotrophic biofilm. To investigate the influence of hydrodynamic conditions on the chemical composition of EPS, a three funnel flow system was used, wherein biofilms were grown at Reynolds numbers of 1000, 2500 and 4000, respectively. 31 and 92 days after inoculation with activated sludge supernatant RM was applied as an additional technique to standard CLSM measurements for a more detailed analysis of the biofilm matrix. Polysaccharide-related Raman bands are in good agreement with the lectin binding analysis from CLSM. For the older biofilm, lectin binding analysis showed no change in the composition of EPS, whereas Raman spectra pointed out a change of EPS composition from predominantly polysaccharides to predominantly (glyco) proteins. For the applied substrate condition no significant influence of the Reynolds number on the chemical properties was observed.

Confocal Laser Scanning Microscopy, a New In Vivo Diagnostic Tool for Schistosomiasis

PubMed Central

Holtfreter, Martha Charlotte; Nohr-Łuczak, Constanze; Guthoff, Rudolf Friedrich; Reisinger, Emil Christian

2012-01-01

Background The gold standard for the diagnosis of schistosomiasis is the detection of the parasite's characteristic eggs in urine, stool, or rectal and bladder biopsy specimens. Direct detection of eggs is difficult and not always possible in patients with low egg-shedding rates. Confocal laser scanning microscopy (CLSM) permits non-invasive cell imaging in vivo and is an established way of obtaining high-resolution images and 3-dimensional reconstructions. Recently, CLSM was shown to be a suitable method to visualize Schistosoma mansoni eggs within the mucosa of dissected mouse gut. In this case, we evaluated the suitability of CLSM to detect eggs of Schistosoma haematobium in a patient with urinary schistosomiasis and low egg-shedding rates. Methodology/Principal Findings The confocal laser scanning microscope used in this study was based on a scanning laser system for imaging the retina of a living eye, the Heidelberg Retina Tomograph II, in combination with a lens system (image modality). Standard light cystoscopy was performed using a rigid cystoscope under general anaesthesia. The CLSM endoscope was then passed through the working channel of the rigid cystoscope. The mucosal tissue of the bladder was scanned using CLSM. Schistoma haematobium eggs appeared as bright structures, with the characteristic egg shape and typical terminal spine. Conclusion/Significance We were able to detect schistosomal eggs in the urothelium of a patient with urinary schistosomiasis. Thus, CLSM may be a suitable tool for the diagnosis of schistosomiasis in humans, especially in cases where standard diagnostic tools are not suitable. PMID:22529947

Confocal laser feedback tomography for skin cancer detection

PubMed Central

Mowla, Alireza; Du, Benjamin Wensheng; Taimre, Thomas; Bertling, Karl; Wilson, Stephen; Soyer, H. Peter; Rakić, Aleksandar D.

2017-01-01

Tomographic imaging of soft tissue such as skin has a potential role in cancer detection. The penetration of infrared wavelengths makes a confocal approach based on laser feedback interferometry feasible. We present a compact system using a semiconductor laser as both transmitter and receiver. Numerical and physical models based on the known optical properties of keratinocyte cancers were developed. We validated the technique on three phantoms containing macro-structural changes in optical properties. Experimental results were in agreement with numerical simulations and structural changes were evident which would permit discrimination of healthy tissue and tumour. Furthermore, cancer type discrimination was also able to be visualized using this imaging technique. PMID:28966845

Confocal laser feedback tomography for skin cancer detection.

PubMed

Mowla, Alireza; Du, Benjamin Wensheng; Taimre, Thomas; Bertling, Karl; Wilson, Stephen; Soyer, H Peter; Rakić, Aleksandar D

2017-09-01

Tomographic imaging of soft tissue such as skin has a potential role in cancer detection. The penetration of infrared wavelengths makes a confocal approach based on laser feedback interferometry feasible. We present a compact system using a semiconductor laser as both transmitter and receiver. Numerical and physical models based on the known optical properties of keratinocyte cancers were developed. We validated the technique on three phantoms containing macro-structural changes in optical properties. Experimental results were in agreement with numerical simulations and structural changes were evident which would permit discrimination of healthy tissue and tumour. Furthermore, cancer type discrimination was also able to be visualized using this imaging technique.

Mapping owl's eye cells of patients with cytomegalovirus corneal endotheliitis using in vivo laser confocal microscopy.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Sugiyama, Kazuhisa

2013-01-01

To produce a two-dimensional reconstruction map of owl's eye cells using in vivo laser confocal microscopy in patients with cytomegalovirus (CMV) corneal endotheliitis, and to demonstrate any association between owl's eye cells and coin-shaped lesions observed with slit-lamp biomicroscopy. Two patients (75- and 77-year-old men) with polymerase chain reaction-proven CMV corneal endotheliitis were evaluated in this study. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Images of owl's eye cells in the endothelial cell layer were arranged and mapped into subconfluent montages. Montage images of owl's eye cells were then superimposed on a slit-lamp photo of the corresponding coin-shaped lesion. Degree of concordance between the confocal microscopic images and slit-lamp photos was evaluated. In both eyes, a two-dimensional reconstruction map of the owl's eye cells was created by computer software using acquired confocal images; the maps showed circular patterns. Superimposing montage images of owl's eye cells onto the photos of a coin-shaped lesion showed good concordance in the two eyes. This study suggests that there is an association between owl's eye cells observed by confocal microscopy and coin-shaped lesions observed by slit-lamp biomicroscopy in patients with CMV corneal endotheliitis. The use of in vivo laser confocal microscopy may provide clues as to the underlying causes of CMV corneal endotheliitis.

A combined confocal and magnetic resonance microscope for biological studies

NASA Astrophysics Data System (ADS)

Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Holtom, Gary R.; Hopkins, Derek F.; Parkinson, Christopher I.; Weber, Thomas J.; Wind, Robert A.

2002-12-01

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of cell function in health and disease, particularly when results acquired with different methodologies can be correlated in time and space. In this article, a novel microscope is described for studying live cells simultaneously with both confocal scanning laser fluorescence optical microscopy and magnetic resonance microscopy. The various design considerations necessary for integrating these two complementary techniques are discussed, the layout and specifications of the instrument are given, and examples of confocal and magnetic resonance images of large frog cells and model tumor spheroids obtained with the compound microscope are presented.

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...

Ultrasonically synthesized organic liquid-filled chitosan microcapsules: part 2: characterization using AFM (atomic force microscopy) and combined AFM-confocal laser scanning fluorescence microscopy.

PubMed

Mettu, Srinivas; Ye, Qianyu; Zhou, Meifang; Dagastine, Raymond; Ashokkumar, Muthupandian

2018-04-25

Atomic Force Microscopy (AFM) is used to measure the stiffness and Young's modulus of individual microcapsules that have a chitosan cross-linked shell encapsulating tetradecane. The oil filled microcapsules were prepared using a one pot synthesis via ultrasonic emulsification of tetradecane and crosslinking of the chitosan shell in aqueous solutions of acetic acid. The concentration of acetic acid in aqueous solutions of chitosan was varied from 0.2% to 25% v/v. The effect of acetic acid concentration and size of the individual microcapsules on the strength was probed. The deformations and forces required to rupture the microcapsules were also measured. Three dimensional deformations of microcapsules under large applied loads were obtained by the combination of Laser Scanning Confocal Microscopy (LSCM) with Atomic Force Microscopy (AFM). The stiffness, and hence the modulus, of the microcapsules was found to decrease with an increase in size with the average stiffness ranging from 82 to 111 mN m-1 and average Young's modulus ranging from 0.4 to 6.5 MPa. The forces required to rupture the microcapsules varied from 150 to 250 nN with deformations of the microcapsules up to 62 to 110% relative to their radius, respectively. Three dimensional images obtained using laser scanning confocal microscopy showed that the microcapsules retained their structure and shape after being subjected to large deformations and subsequent removal of the loads. Based on the above observations, the oil filled chitosan crosslinked microcapsules are an ideal choice for use in the food and pharmaceutical industries as they would be able to withstand the process conditions encountered.

Laser-induced cartilage damage: an ex-vivo model using confocal microscopy

NASA Astrophysics Data System (ADS)

Frenz, Martin; Zueger, Benno J.; Monin, D.; Weiler, C.; Mainil-Varlet, P. M.; Weber, Heinz P.; Schaffner, Thomas

1999-06-01

Although there is an increasing popularity of lasers in orthopedic surgery, there is a growing concern about negative side effects of this therapy e.g. prolonged restitution time, radiation damage to adjacent cartilage or depth effects like bone necrosis. Despite case reports and experimental investigations over the last few years little is known about the extent of acute cartilage damage induced by different lasers types and energies. Histological examination offers only limited insights in cell viability and metabolism. Ho:YAG and Er:YAG lasers emitting at 2.1 micrometer and 2.94 micrometer, respectively, are ideally suited for tissue treatment because these wavelengths are strongly absorbed in water. The Purpose of the present study is to evaluate the effect of laser type and energy on chondrocyte viability in an ex vivo model. Free running Er:YAG (E equals 100 and 150 mJ) and Ho:YAG (E equals 500 and 800 mJ) lasers were used at different energy levels using a fixed pulse length of 400 microseconds. The energy was delivered at 8 Hz through optical fibers. Fresh bovine hyaline cartilage samples were mounted in a water bath at room temperature and the fiber was positioned at 30 degree and 180 degree angles relative to the tissue surface. After laser irradiation the samples were assessed by a life-dead cell viability test using a confocal microscope and by standard histology. Thermal damage was much deeper with Ho:YAG (up to 1800 micrometer) than with the Er:YAG laser (up to 70 micrometer). The cell viability test revealed a damage zone about twice the one determined by standard histology. Confocal microscopy is a powerful tool for assessing changes in tissue structure after laser treatment. In addition this technique allows to quantify these alterations without necessitating time consuming and expensive animal experiments.

CONFOCAL LASER SCANNING MICROSCOPY OF APOPTOSIS IN WHOLE MOUSE AND RAT OVARIES

EPA Science Inventory

Confocal Laser Scanning Microscopy of Apoptosis in Whole Mouse and Rat Ovaries. Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research ...

Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

NASA Astrophysics Data System (ADS)

Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

2016-03-01

Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. 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 cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

Confocal laser endomicroscopy in the "in vivo" histological diagnosis of the gastrointestinal tract.

PubMed

De Palma, Giovanni D

2009-12-14

Recent technological advances in miniaturization have allowed for a confocal scanning microscope to be integrated into a conventional flexible endoscope, or into trans-endoscopic probes, a technique now known as confocal endomicroscopy or confocal laser endomicroscopy. This newly-developed technology has enabled endoscopists to collect real-time in vivo histological images or "virtual biopsies" of the gastrointestinal mucosa during endoscopy, and has stimulated significant interest in the application of this technique in clinical gastroenterology. This review aims to evaluate the current data on the technical aspects and the utility of this new technology in clinical gastroenterology and its potential impact in the future, particularly in the screening or surveillance of gastrointestinal neoplasia.

Diffraction-Unlimited Fluorescence Imaging with an EasySTED Retrofitted Confocal Microscope.

PubMed

Klauss, André; Hille, Carsten

2017-01-01

The easySTED technology provides the means to retrofit a confocal microscope to a diffraction-unlimited stimulated emission depletion (STED) microscope.Although commercial STED systems are available today, for many users of confocal laser scanning microscopes the option of retrofitting their confocal system to a STED system ready for diffraction-unlimited imaging may present an attractive option. The easySTED principle allowing for a joint beam path of excitation and depletion light promises some advantages concerning technical complexity and alignment effort for such an STED upgrade. In the one beam path design of easySTED the use of a common laser source, either a supercontinuum source or two separate lasers coupled into the same single-mode fiber, becomes feasible. The alignment of the focal light distribution of the STED beam relative to that of the excitation beam in all three spatial dimensions is therefore omitted respectively reduced to coupling the STED laser into the common single-mode fiber. Thus, only minor modifications need to be applied to the beam path in the confocal microscope to be upgraded. Those comprise adding polarization control elements and the easySTED waveplate, and adapting the beamsplitter to the excitation/STED wavelength combination.

Application of laser differential confocal technique in back vertex power measurement for phoropters

NASA Astrophysics Data System (ADS)

Li, Fei; Li, Lin; Ding, Xiang; Liu, Wenli

2012-10-01

A phoropter is one of the most popular ophthalmic instruments used in optometry and the back vertex power (BVP) is one of the most important parameters to evaluate the refraction characteristics of a phoropter. In this paper, a new laser differential confocal vertex-power measurement method which takes advantage of outstanding focusing ability of laser differential confocal (LDC) system is proposed for measuring the BVP of phoropters. A vertex power measurement system is built up. Experimental results are presented and some influence factor is analyzed. It is demonstrated that the method based on LDC technique has higher measurement precision and stronger environmental anti-interference capability compared to existing methods. Theoretical analysis and experimental results indicate that the measurement error of the method is about 0.02m-1.

3-D reconstruction of neurons from multichannel confocal laser scanning image series.

PubMed

Wouterlood, Floris G

2014-04-10

A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out-of-focus information. Scanning of a specimen, with stepwise axial (Z-) movement of the stage in between each scan, produces Z-series of confocal images of a tissue volume, which then can be used to 3-D reconstruct structures of interest. The operator first configures separate channels (e.g., laser, filters, and detector settings) for each applied fluorochrome and then acquires Z-series of confocal images: one series per channel. Channel signal separation is extremely important. Measures to avoid bleaching are vital. Post-acquisition deconvolution of the image series is often performed to increase resolution before 3-D reconstruction takes place. In the 3-D reconstruction programs described in this unit, reconstructions can be inspected in real time from any viewing angle. By altering viewing angles and by switching channels off and on, the spatial relationships of 3-D-reconstructed structures with respect to structures visualized in other channels can be studied. Since each brand of CLSM, computer program, and 3-D reconstruction package has its own proprietary set of procedures, a general approach is provided in this protocol wherever possible. Copyright © 2014 John Wiley & Sons, Inc.

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.

Passport examination by a confocal-type laser profile microscope.

PubMed

Sugawara, Shigeru

2008-06-10

The author proposes a nondestructive and highly precise method of measuring the thickness of a film pasted on a passport using a confocal-type laser profile microscope. The effectiveness of this method in passport examination is demonstrated. A confocal-type laser profile microscope is used to create profiles of the film surface and film-paper interface; these profiles are used to calculate the film thickness by employing an algorithm developed by the author. The film thicknesses of the passport samples--35 genuine and 80 counterfeit Japanese passports--are measured nondestructively. The intra-sample standard deviation of the film thicknesses of the genuine and counterfeit Japanese passports was of the order of 1 microm The intersample standard deviations of the film thicknesses of passports forged using the same tools and techniques are expected to be of the order of 1 microm. The thickness values of the films on the machine-readable genuine passports ranged between 31.95 microm and 36.95 microm. The likelihood ratio of this method in the authentication of machine-readable Japanese genuine passports is 11.7. Therefore, this method is effective for the authentification of genuine passports. Since the distribution of the film thickness of all forged passports was considerably larger than the accuracy of this method, this method is considered effective also for revealing the relation among the forged passports and acquiring proof of the crime.

How the confocal laser scanning microscope entered biological research.

PubMed

Amos, W B; White, J G

2003-09-01

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.

Spectral imaging technique for retinal perfusion detection using confocal scanning laser ophthalmoscopy

NASA Astrophysics Data System (ADS)

Rasta, Seyed Hossein; Manivannan, Ayyakkannu; Sharp, Peter F.

2012-11-01

To evaluate retinal perfusion in the human eye, a dual-wavelength confocal scanning laser ophthalmoscope (cSLO) was developed that provides spectral imaging of the fundus using a combination of red (670 nm) and near-infrared (810 nm) wavelengths. The image of the ocular fundus was analyzed to find out if quantitative measurements of the reflectivity of tissue permit assessment of the oxygen perfusion of tissue. We explored problems that affect the reproducibility of patient measurements such as non-uniformity errors on the image. For the first time, an image processing technique was designed and used to minimize the errors of oxygen saturation measurements by illumination correction in retina wide field by increasing SNR. Retinal images were taken from healthy and diabetic retinopathy eyes using the cSLO with a confocal aperture of 100 μm. The ratio image (RI) of red/IR, as oxygen saturation (SO2) index, was calculated for normal eyes. The image correction technique improved the reproducibility of the measurements. Average RI intensity variation of healthy retina tissue was determined within a range of about 5.5%. The capability of the new technique to discriminate oxygenation levels of retinal artery and vein was successfully demonstrated and showed good promise in the diagnosis of the perfused retina.

A data processing method based on tracking light spot for the laser differential confocal component parameters measurement system

NASA Astrophysics Data System (ADS)

Shao, Rongjun; Qiu, Lirong; Yang, Jiamiao; Zhao, Weiqian; Zhang, Xin

2013-12-01

We have proposed the component parameters measuring method based on the differential confocal focusing theory. In order to improve the positioning precision of the laser differential confocal component parameters measurement system (LDDCPMS), the paper provides a data processing method based on tracking light spot. To reduce the error caused by the light point moving in collecting the axial intensity signal, the image centroiding algorithm is used to find and track the center of Airy disk of the images collected by the laser differential confocal system. For weakening the influence of higher harmonic noises during the measurement, Gaussian filter is used to process the axial intensity signal. Ultimately the zero point corresponding to the focus of the objective in a differential confocal system is achieved by linear fitting for the differential confocal axial intensity data. Preliminary experiments indicate that the method based on tracking light spot can accurately collect the axial intensity response signal of the virtual pinhole, and improve the anti-interference ability of system. Thus it improves the system positioning accuracy.

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the "map" area, mainly in the basal epithelial cell layer. In "fingerprint" lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities.

Toward real-time virtual biopsy of oral lesions using confocal laser endomicroscopy interfaced with embedded computing.

PubMed

Thong, Patricia S P; Tandjung, Stephanus S; Movania, Muhammad Mobeen; Chiew, Wei-Ming; Olivo, Malini; Bhuvaneswari, Ramaswamy; Seah, Hock-Soon; Lin, Feng; Qian, Kemao; Soo, Khee-Chee

2012-05-01

Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies.

PubMed

Parker, I; Callamaras, N; Wier, W G

1997-06-01

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca2+ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record line-scans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.

Laser scanning confocal microscope with programmable amplitude, phase, and polarization of the illumination beam.

PubMed

Boruah, B R; Neil, M A A

2009-01-01

We describe the design and construction of a laser scanning confocal microscope with programmable beam forming optics. The amplitude, phase, and polarization of the laser beam used in the microscope can be controlled in real time with the help of a liquid crystal spatial light modulator, acting as a computer generated hologram, in conjunction with a polarizing beam splitter and two right angled prisms assembly. Two scan mirrors, comprising an on-axis fast moving scan mirror for line scanning and an off-axis slow moving scan mirror for frame scanning, configured in a way to minimize the movement of the scanned beam over the pupil plane of the microscope objective, form the XY scan unit. The confocal system, that incorporates the programmable beam forming unit and the scan unit, has been implemented to image in both reflected and fluorescence light from the specimen. Efficiency of the system to programmably generate custom defined vector beams has been demonstrated by generating a bottle structured focal volume, which in fact is the overlap of two cross polarized beams, that can simultaneously improve both the lateral and axial resolutions if used as the de-excitation beam in a stimulated emission depletion confocal microscope.

Low-power laser effects at the single-cell level: a confocal microscopy study

NASA Astrophysics Data System (ADS)

Alexandratou, Eleni; Yova, Dido M.; Atlamazoglou, Vassilis; Handris, Panagiotis; Kletsas, Dimitris; Loukas, Spyros

2000-11-01

Confocal microscopy was used for irradiation and observation of the same area of interest, allowing the imaging of low power laser effects in subcellular components and functions, at the single cell level. Coverslips cultures of human fetal foreskin fibroblasts (HFFF2) were placed in a small incubation chamber for in vivo microscopic observation. Cells were stimulated by the 647 nm line of the Argon- Krypton laser of the confocal microscope (0.1 mW/cm2). Membrane permeability, mitochondrial membrane potential ((delta) Psim), intracellular pHi, calcium alterations and nuclear chromatin accessibility were monitored, at different times after irradiation, using specific fluorescent vital probes. Images were stored to the computer and quantitative evaluation was performed using image- processing software. After irradiation, influx and efflux of the appropriate dyes monitored changes in cell membrane permeability. Laser irradiation caused alkalizatoin of the cytosolic pHi and increase of the mitochondrial membrane potential ((delta) Psim). Temporary global Ca2+ responses were also observed. No such effects were noted in microscopic fields other than the irradiated ones. No toxic effects were observed, during time course of the experiment.

Laser scanning in vivo confocal microscopy of the normal human corneoscleral limbus.

PubMed

Patel, Dipika V; Sherwin, Trevor; McGhee, Charles N J

2006-07-01

To elucidate the structure of the human corneoscleral limbus by in vivo laser scanning confocal microscopy and to correlate limbal epithelial dimensions and density with the central epithelium and in relation to age. Fifty adult subjects were recruited into one of two age groups: younger (age<45 years) and older (age>or=45 years). Fifty left eyes of these 50 healthy subjects were examined by laser scanning in vivo confocal microscopy, to assess the basal epithelium of the central cornea and inferior limbus. Mean epithelial cell diameter, area, and density were calculated for the central basal epithelium, limbus-corneal basal epithelium, and limbus-palisade epithelium. Data were analyzed in relation to the two age groups, group A, 30+/-6 years (n=25; mean+/-SD), and group B, 60+/-11 years (n=25; P<0.01). Mean epithelial density in the limbus-cornea and limbus-palisade regions decreased significantly with age: limbus-cornea group A=7253+/-1077 cells/mm2 group B=6614+/-987 cells/mm2, P=0.03; limbus palisade group A=5409+/-799 cells/mm2, group B=5055+/-722 cells/mm2, P=0.03). Central corneal epithelial density did not change with age: group A=6162+/-503 cells/mm2, group B=6362+/-614 cells/mm2, P=0.08. Mean epithelial density was greatest at the limbus-cornea (7010+/-1081 cells/mm2) and lowest at the limbus-palisades (5289+/-847 cells/mm2). The mean width of palisade ridges was 25.0+/-6.3 microm. This is the first study to image clearly the living human corneal limbus by laser scanning in vivo confocal microscopy and to demonstrate quantitative changes in the basal epithelium with age.

The fractional laser-induced coagulation zone characterized over time by laser scanning confocal microscopy-A proof of concept study.

PubMed

Banzhaf, Christina A; Lin, Lynlee L; Dang, Nhung; Freeman, Michael; Haedersdal, Merete; Prow, Tarl W

2018-01-01

Ablative fractional laser (AFXL) is an acknowledged technique to increase uptake of topical agents in skin. Micro thermal ablation zones (MAZs) consist of ablated vertical channels surrounded by a coagulation zone (CZ). Laser scanning confocal microscopy (LSCM) images individual MAZs at 733 nm (reflectance confocal microscopy (RCM)). Further, LSCM can image sodium fluorescein (NaF) fluorescence with 488 nm excitation (fluorescence confocal microcopy (FCM)), a small hydrophilic test molecule (370 MW, log P -1.52), which may simulate uptake, bio-distribution and kinetics of small hydrophilic drugs. To explore LSCM for combined investigations of CZ thickness and uptake, bio-distribution and kinetics of NaF in AFXL-exposed skin. Excised human abdominal skin samples were exposed to AFXL (15 mJ/microbeam, 2% density) and NaF gel (1000 μg/ml, 10 μl/cm2) in six repetitions, including untreated control samples. CZ thickness and spatiotemporal fluorescence intensities (FI) were quantified up to four hours after NaF application by RCM and FCM. Test sites were scanned to a depth of 200 μm, quantifying thickness of skin compartments (stratum corneum, epidermis, upper dermis), individual CZ thicknesses and FI in CZ and surrounding skin. RCM images established skin morphology to a depth of 200 μm. The CZ thickness measurements were feasible to a depth of 50 μm, and remained unchanged over time at 50 μm (P > 0.5). FI were detected to a depth of 160 μm and remained constant in CZ up to four hours after NaF application (15 minutes: 79 AU (73-92 AU), 60 minutes: 72 AU (58-82 AU), four hours: 78 AU (71-90 AU), P > 0.1). In surrounding skin, FI increased significantly over time, but remained lower than FI in CZ (15 minutes: 21 AU (17-22 AU), 60 minutes: 21 AU (19-26 AU), four hours: 42 (31- 48 AU), P = 0.03). AFXL-processed skin generated higher FI compared to non-laser processed skin in epidermis and upper dermis at 60 minutes and four hours

Morphological and ultrastructural characterization of ionoregulatory cells in the teleost Oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique.

PubMed

Fridman, Sophie; Rana, Krishen J; Bron, James E

2013-10-01

Aspects of ionoregulatory or mitochondria-rich cell (MRC) differentiation and adaptation in Nile tilapia yolk-sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole-mount Nile tilapia larvae using anti- Na⁺/K⁺-ATPase as a primary antibody and Fluoronanogold™ (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk-sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺-ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level. Copyright © 2013 Wiley Periodicals, Inc.

Confocal laser scanning microscopy to estimate nanoparticles' human skin penetration in vitro.

PubMed

Zou, Ying; Celli, Anna; Zhu, Hanjiang; Elmahdy, Akram; Cao, Yachao; Hui, Xiaoying; Maibach, Howard

2017-01-01

With rapid development of nanotechnology, there is increasing interest in nanoparticle (NP) application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration. Three different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO) and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy. NPs were localized in the stratum corneum (SC) and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not. Except with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human "viable" epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested.

Three-dimensional imaging of porous media using confocal laser scanning microscopy.

PubMed

Shah, S M; Crawshaw, J P; Boek, E S

2017-02-01

In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO 2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Probe-based confocal laser endomicroscopy (pCLE) - a new imaging technique for in situ localization of spermatozoa.

PubMed

Trottmann, Matthias; Stepp, Herbert; Sroka, Ronald; Heide, Michael; Liedl, Bernhard; Reese, Sven; Becker, Armin J; Stief, Christian G; Kölle, Sabine

2015-05-01

In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe-based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non-invasive, real-time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real-time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively. Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe-based laser endomicroscopy (pCLE, right) using Pro Flex(TM) UltraMini O after staining with acriflavine. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy

PubMed Central

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

Background: The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Methods: Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Results: Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the “map” area, mainly in the basal epithelial cell layer. In “fingerprint” lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. Conclusion: HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities. PMID:22888214

Application of confocal laser microscopy for monitoring mesh implants in herniology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zakharov, V P; Belokonev, V I; Bratchenko, I A

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)

High-power direct green laser oscillation of 598 mW in Pr(3+)-doped waterproof fluoroaluminate glass fiber excited by two-polarization-combined GaN laser diodes.

PubMed

Nakanishi, Jun; Horiuchi, Yuya; Yamada, Tsuyoshi; Ishii, Osamu; Yamazaki, Masaaki; Yoshida, Minoru; Fujimoto, Yasushi

2011-05-15

We demonstrated a high-power and highly efficient Pr-doped waterproof fluoride glass fiber laser at 522.2 nm excited by two-polarization-combined GaN laser diodes and achieved a subwatt output power of 598 mW and slope efficiency of 43.0%. This system will enable us to make a vivid laser display, a photocoagulation laser for eye surgery, a color confocal scanning laser microscope, and an effective laser for material processing. Direct visible ultrashort pulse generation is also expected. © 2011 Optical Society of America

Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

PubMed Central

Castellano-Muñoz, Manuel; Peng, Anthony Wei; Salles, Felipe T.; Ricci, Anthony J.

2013-01-01

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality. PMID:22831554

Automatic analysis for neuron by confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Satou, Kouhei; Aoki, Yoshimitsu; Mataga, Nobuko; Hensh, Takao K.; Taki, Katuhiko

2005-12-01

The aim of this study is to develop a system that recognizes both the macro- and microscopic configurations of nerve cells and automatically performs the necessary 3-D measurements and functional classification of spines. The acquisition of 3-D images of cranial nerves has been enabled by the use of a confocal laser scanning microscope, although the highly accurate 3-D measurements of the microscopic structures of cranial nerves and their classification based on their configurations have not yet been accomplished. In this study, in order to obtain highly accurate measurements of the microscopic structures of cranial nerves, existing positions of spines were predicted by the 2-D image processing of tomographic images. Next, based on the positions that were predicted on the 2-D images, the positions and configurations of the spines were determined more accurately by 3-D image processing of the volume data. We report the successful construction of an automatic analysis system that uses a coarse-to-fine technique to analyze the microscopic structures of cranial nerves with high speed and accuracy by combining 2-D and 3-D image analyses.

High-precision radius automatic measurement using laser differential confocal technology

NASA Astrophysics Data System (ADS)

Jiang, Hongwei; Zhao, Weiqian; Yang, Jiamiao; Guo, Yongkui; Xiao, Yang

2015-02-01

A high precision radius automatic measurement method using laser differential confocal technology is proposed. Based on the property of an axial intensity curve that the null point precisely corresponds to the focus of the objective and the bipolar property, the method uses the composite PID (proportional-integral-derivative) control to ensure the steady movement of the motor for process of quick-trigger scanning, and uses least-squares linear fitting to obtain the position of the cat-eye and confocal positions, then calculates the radius of curvature of lens. By setting the number of measure times, precision auto-repeat measurement of the radius of curvature is achieved. The experiment indicates that the method has the measurement accuracy of better than 2 ppm, and the measuring repeatability is better than 0.05 μm. In comparison with the existing manual-single measurement, this method has a high measurement precision, a strong environment anti-interference capability, a better measuring repeatability which is only tenth of former's.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

PubMed

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

Confocal laser scanning microscopy to estimate nanoparticles’ human skin penetration in vitro

PubMed Central

Elmahdy, Akram; Cao, Yachao; Hui, Xiaoying; Maibach, Howard

2017-01-01

Objective With rapid development of nanotechnology, there is increasing interest in nanoparticle (NP) application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration. Methods Three different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO) and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy. Results NPs were localized in the stratum corneum (SC) and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not. Conclusion Except with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human “viable” epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested. PMID:29184403

Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

PubMed Central

Zehri, Aqib H.; Ramey, Wyatt; Georges, Joseph F.; Mooney, Michael A.; Martirosyan, Nikolay L.; Preul, Mark C.; Nakaji, Peter

2014-01-01

Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid) with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS) microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM), two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery. PMID:24872922

Laser confocal measurement system for curvature radius of lenses based on grating ruler

NASA Astrophysics Data System (ADS)

Tian, Jiwei; Wang, Yun; Zhou, Nan; Zhao, Weirui; Zhao, Weiqian

2015-02-01

In the modern optical measurement field, the radius of curvature (ROC) is one of the fundamental parameters of optical lens. Its measurement accuracy directly affects the other optical parameters, such as focal length, aberration and so on, which significantly affect the overall performance of the optical system. To meet the demand of measurement instruments for radius of curvature (ROC) with high accuracy in the market, we develop a laser confocal radius measurement system with grating ruler. The system uses the peak point of the confocal intensity curve to precisely identify the cat-eye and confocal positions and then measure the distance between these two positions by using the grating ruler, thereby achieving the high-precision measurement for the ROC. The system has advantages of high focusing sensitivity and anti-environment disturbance ability. And the preliminary theoretical analysis and experiments show that the measuring repeatability can be up to 0.8 um, which can provide an effective way for the accurate measurement of ROC.

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy

PubMed Central

Siegel, Nisan; Brooker, Gary

2014-01-01

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called “CINCH”. PMID:25321701

ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

PubMed Central

Hng, Keng Imm; Dormann, Dirk

2013-01-01

Laser scanning confocal microscopy has become an invaluable tool in biomedical research but regular quality testing is vital to maintain the system’s performance for diagnostic and research purposes. Although many methods have been devised over the years to characterise specific aspects of a confocal microscope like measuring the optical point spread function or the field illumination, only very few analysis tools are available. Our aim was to develop a comprehensive quality assurance framework ranging from image acquisition to automated analysis and documentation. We created standardised test data to assess the performance of the lasers, the objective lenses and other key components required for optimum confocal operation. The ConfocalCheck software presented here analyses the data fully automatically. It creates numerous visual outputs indicating potential issues requiring further investigation. By storing results in a web browser compatible file format the software greatly simplifies record keeping allowing the operator to quickly compare old and new data and to spot developing trends. We demonstrate that the systematic monitoring of confocal performance is essential in a core facility environment and how the quantitative measurements obtained can be used for the detailed characterisation of system components as well as for comparisons across multiple instruments. PMID:24224017

Epiphany sealer penetration into dentinal tubules: Confocal laser scanning microscopic study.

PubMed

Ravi, S V; Nageswar, Rao; Swapna, Honwad; Sreekant, Puthalath; Ranjith, Madhavan; Mahidhar, Surabhi

2014-03-01

The aim of the following study was to evaluate the percentage and average depth of epiphany sealer penetration into dentinal tubules among the coronal, middle and apical thirds of the root using the confocal laser scanning microscopy (CLSM). A total of 10 maxillary central incisors were prepared and obturated with Resilon-Epiphany system. Sealer was mixed with fluorescent rhodamine B isothiyocyanate dye for visibility under confocal microscope. Teeth were cross-sectioned into coronal, middle and apical sections-2 mm thick. Sections were observed under CLSM. Images were analyzed for percentage and average depth of sealer penetration into dentinal tubules using the lasso tool in Adobe Photoshop CS3 (Adobe systems incorporated, San jose, CA) and laser scanning microscopy (LSM 5) image analyzer. One-way analysis of variance with Student Neuman Keuls post hoc tests, Kruskal-Wallis test and Wilcoxon signed-rank post hoc tests. The results showed that a higher percentage of sealer penetration in coronal section-89.23%, followed by middle section-84.19% and the apical section-64.9%. Average depth of sealer penetration for coronal section was 526.02 μm, middle-385.26 μm and apical-193.49 μm. Study concluded that there was higher epiphany sealer penetration seen in coronal followed by middle and least at apical third of the roots.

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.

In vivo laser confocal microscopy findings of radial keratoneuritis in patients with early stage Acanthamoeba keratitis.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Yamazaki, Natsuko; Ishibashi, Yasuhisa; Oikawa, Yosaburo; Tokoro, Masaharu; Sugiyama, Kazuhisa

2013-07-01

To investigate in vivo corneal changes of keratoneuritis in early stage Acanthamoeba keratitis (AK) using in vivo laser confocal microscopy. Single-center, prospective, clinical study. Thirteen eyes (12 patients; 5 men and 7 women; mean age ± standard deviation, 22.3 ± 4.2 years) with keratoneuritis resulting from early stage AK were included in this study. In vivo laser confocal microscopy was performed, paying special attention to keratoneuritis. Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of abnormal cells and deposits. In all patients, Acanthamoeba cysts were observed clearly in the basal epithelial cell layer as highly reflective round particles with a diameter of 10 to 20 μm. Bowman's layer infiltration of Acanthamoeba cysts was observed in only 1 case, and no cases showed stromal or nerve infiltration of Acanthamoeba cysts. In the stroma, all cases showed highly reflective activated keratocytes forming a honeycomb pattern; these changes were significant around the keratoneuritis. Infiltration of inflammatory cells, possibly polymorphonuclear cells, was observed along with keratocyte bodies in all cases. Numerous highly reflective spindle-shaped materials were observed around the keratoneuritis. Most notably, highly reflective patchy lesions were observed around the keratoneuritis in 11 cases (84.6%). Inflammatory cells also were observed in the endothelial cell layer in 4 cases (30.8%). In vivo laser confocal microscopy identified consistent corneal abnormalities around keratoneuritis in early stage AK patients, of which highly reflective patchy lesions may be characteristic of keratoneuritis. Further morphologic studies of corneas with early stage AK in a larger number of patients may elucidate the clinical significance of radial keratoneuritis and may help us to understand the interaction between Acanthamoeba organisms and host corneal cells or nerves. Copyright © 2013 American

Evaluation of conjunctival inflammatory status by confocal scanning laser microscopy and conjunctival brush cytology in patients with atopic keratoconjunctivitis (AKC)

PubMed Central

Wakamatsu, Tais Hitomi; Okada, Naoko; Kojima, Takashi; Matsumoto, Yukihiro; Ibrahim, Osama M.A.; Adan, Enrique Sato; Fukagawa, Kazumi; Katakami, Chikako; Tsubota, Kazuo; Shimazaki, Jun; Fujishima, Hiroshi

2009-01-01

Purpose To elucidate the status of the conjunctival inflammation in atopic keratoconjunctivitis (AKC) using laser scanning confocal microscopy and compare the relevant findings with conjunctival brush cytology in a prospective controlled study. Methods Twenty eyes from 20 AKC patients as well as 16 eyes from 16 age and sex matched normal subjects were studied. The subjects underwent tear film break-up time (BUT), fluorescein and Rose Bengal staining of the ocular surface, conjunctival confocal microscopy, Schirmer test, and brush cytology. Brush cytology specimens and in vivo confocal microscopy scans underwent evaluation for inflammatory cell densities. Results Brush cytology specimens and in vivo confocal microscopy scans from AKC patients revealed significantly higher numbers of inflammatory cells (p<0.05). Conjunctival inflammatory cell density showed a negative correlation with tear stability and a positive correlation with vital staining scores and conjunctival injection grades. The extent of conjunctival inflammation assessed by in vivo confocal microscopy showed a strong positive linear correlation with the inflammation status evaluated by brush cytology. The corneal inflammatory cell density assessed by in vivo confocal microscopy showed a significant negative correlation with tear stability and a positive linear correlation with corneal fluorescein staining. Conclusions Confocal scanning laser microscopy is an efficient, noninvasive, and a promising tool for the quantitative assessment of conjunctival inflammation, a parameter of this new technology which correlated well with subjective and objective ocular surface clinical findings. PMID:19693288

Characterization of particle deformation during compression measured by confocal laser scanning microscopy.

PubMed

Guo, H X; Heinämäki, J; Yliruusi, J

1999-09-20

Direct compression of riboflavin sodium phosphate tablets was studied by confocal laser scanning microscopy (CLSM). The technique is non-invasive and generates three-dimensional (3D) images. Tablets of 1% riboflavin sodium phosphate with two grades of microcrystalline cellulose (MCC) were individually compressed at compression forces of 1.0 and 26.8 kN. The behaviour and deformation of drug particles on the upper and lower surfaces of the tablets were studied under compression forces. Even at the lower compression force, distinct recrystallized areas in the riboflavin sodium phosphate particles were observed in both Avicel PH-101 and Avicel PH-102 tablets. At the higher compression force, the recrystallization of riboflavin sodium phosphate was more extensive on the upper surface of the Avicel PH-102 tablet than the Avicel PH-101 tablet. The plastic deformation properties of both MCC grades reduced the fragmentation of riboflavin sodium phosphate particles. When compressed with MCC, riboflavin sodium phosphate behaved as a plastic material. The riboflavin sodium phosphate particles were more tightly bound on the upper surface of the tablet than on the lower surface, and this could also be clearly distinguished by CLSM. Drug deformation could not be visualized by other techniques. Confocal laser scanning microscopy provides valuable information on the internal mechanisms of direct compression of tablets.

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...

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.

Improving confocal microscopy with solid-state semiconductor excitation sources

NASA Astrophysics Data System (ADS)

Sivers, Nelson L.

To efficiently excite the fluorescent dyes used in imaging biological samples with a confocal microscope, the wavelengths of the exciting laser must be near the fluorochrome absorption peak. However, this causes imaging problems when the fluorochrome absorption and emission spectra overlap significantly, i.e. have small Stokes shifts, which is the case for most fluorochromes that emit in the red to infrared. As a result, the reflected laser excitation cannot be distinguished from the information-containing fluorescence signal. However, cryogenically cooling the exciting laser diode enabled the laser emission wavelengths to be tuned to shorter wavelengths, decreasing the interference between the laser and the fluorochrome's fluorescence. This reduced the amount of reflected laser light in the confocal image. However, the cooled laser diode's shorter wavelength signal resulted in slightly less efficient fluorochrome excitation. Spectrophotometric analysis showed that as the laser diodes were cooled, their output power increased, which more than compensated for the lower fluorochrome excitation and resulted in significantly more intense fluorescence. Thus, by tuning the laser diode emission wavelengths away from the fluorescence signal, less reflected laser light and more fluorescence information reached the detector, creating images with better signal to noise ratios. Additionally, new, high, luminous flux, light-emitting diodes (LEDs) are now powerful enough to create confocal fluorescence signals comparable to those produced by the traditional laser excitation sources in fluorescence confocal microscopes. The broader LED spectral response effectively excited the fluorochrome, yet was spectrally limited enough for standard filter sets to separate the LED excitation from the fluorochrome fluorescence signal. Spectrophotometric analysis of the excitation and fluorescence spectra of several fluorochromes showed that high-powered, LED-induced fluorescence contained the

A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

PubMed Central

Kang, Minchul; Day, Charles A.; Drake, Kimberly; Kenworthy, Anne K.; DiBenedetto, Emmanuele

2009-01-01

Abstract Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells. PMID:19720039

Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope.

PubMed

Steinbach, Gábor; Pomozi, István; Zsiros, Ottó; Páy, Anikó; Horváth, Gábor V; Garab, Gyozo

2008-03-01

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence-detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity-difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber-laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture. Copyright 2007 International Society for Analytical Cytology.

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM - SEM).

PubMed

Schaudinn, C; Carr, G; Gorur, A; Jaramillo, D; Costerton, J W; Webster, P

2009-08-01

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.

Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier.

PubMed

Ilev, Ilko; Waynant, Ronald; Gannot, Israel; Gandjbakhche, Amir

2007-09-01

A novel fiber-optic confocal approach for ultrahigh depth-resolution (confocal microscope approach that is compatible with a differential confocal microscope technique. To improve the dynamic range of the resolving laser power and to achieve a high resolution in the nanometric range, we have designed a simple apertureless reflection confocal microscope with a highly sensitive single-mode-fiber confocal output. The fiber-optic design is an effective alternative to conventional pinhole-based confocal systems and offers a number of advantages in terms of spatial resolution, flexibility, miniaturization, and scanning potential. Furthermore, the design is compatible with the differential confocal pinhole microscope based on the use of the sharp diffraction-free slope of the axial confocal response curve rather than the area around the maximum of that curve. Combining the advantages of ultrahigh-resolution fiber-optic confocal microscopy, we can work beyond the diffraction barrier in the subwavelength (below 200 nm) nanometric range exploiting confocal nanobioimaging of single cell and intracellular analytes.

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

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

PubMed

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

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.

Multimodal backside imaging of a microcontroller using confocal laser scanning and optical-beam-induced current imaging

NASA Astrophysics Data System (ADS)

Finkeldey, Markus; Göring, Lena; Schellenberg, Falk; Brenner, Carsten; Gerhardt, Nils C.; Hofmann, Martin

2017-02-01

Microscopy imaging with a single technology is usually restricted to a single contrast mechanism. Multimodal imaging is a promising technique to improve the structural information that could be obtained about a device under test (DUT). Due to the different contrast mechanisms of laser scanning microscopy (LSM), confocal laser scanning microscopy (CLSM) and optical beam induced current microscopy (OBICM), a combination could improve the detection of structures in integrated circuits (ICs) and helps to reveal their layout. While OBIC imaging is sensitive to the changes between differently doped areas and to semiconductor-metal transitions, CLSM imaging is mostly sensitive to changes in absorption and reflection. In this work we present the implementation of OBIC imaging into a CLSM. We show first results using industry standard Atmel microcontrollers (MCUs) with a feature size of about 250nm as DUTs. Analyzing these types of microcontrollers helps to improve in the field of side-channel attacks to find hardware Trojans, possible spots for laser fault attacks and for reverse engineering. For the experimental results the DUT is placed on a custom circuit board that allows us to measure the current while imaging it in our in-house built stage scanning microscope using a near infrared (NIR) laser diode as light source. The DUT is thinned and polished, allowing backside imaging through the Si-substrate. We demonstrate the possibilities using this optical setup by evaluating OBIC, LSM and CLSM images above and below the threshold of the laser source.

Clinical significance of owl eye morphologic features by in vivo laser confocal microscopy in patients with cytomegalovirus corneal endotheliitis.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Higashide, Tomomi; Nitta, Koji; Sugiyama, Kazuhisa

2012-03-01

To demonstrate the clinical significance of owl eye morphologic features observed by in vivo laser confocal microscopy in patients with cytomegalovirus (CMV) corneal endotheliitis. Observational case series. participants: Six eyes of 6 patients (6 men; mean age, 73.3 years) with cytomegalovirus corneal endotheliitis diagnosed by clinical manifestations together with polymerase chain reaction from aqueous humor samples. intervention: All patients were examined by slit-lamp biomicroscopy and in vivo laser confocal microscopy. main outcome measures: Clinical manifestations were summarized by reviewing medical records. Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of abnormal cells and deposits. All patients had long histories of anterior uveitis with intraocular pressure elevation, corneal edema with keratic precipitates, and decrease of endothelial cell densities. Coin-shaped lesions were observed by slit lamp only in 1 patient at the first visit and in 2 additional patients at subsequent follow-up. In all patients, confocal microscopy demonstrated reduced subepithelial nerves, subepithelial opacity, increased reflectivity of keratocytes, highly reflective dots, and needle-shaped bodies. Owl eye morphologic features were observed consistently in all patients at the initial visit, and highly reflective round bodies were detected in 5 patients; most notably, these confocal features were reversible after resolution of endotheliitis. Owl eye morphologic features and highly reflective round bodies observed by confocal microscopy may be useful as an adjunct for the noninvasive diagnosis of cytomegalovirus corneal endotheliitis. Reversibility of these features after resolution of endotheliitis may be useful for monitoring the therapeutic effects without multiple anterior chamber tap. Copyright © 2012 Elsevier Inc. All rights reserved.

Plasmon resonance and the imaging of metal-impregnated neurons with the laser scanning confocal microscope

PubMed Central

Thompson, Karen J; Harley, Cynthia M; Barthel, Grant M; Sanders, Mark A; Mesce, Karen A

2015-01-01

The staining of neurons with silver began in the 1800s, but until now the great resolving power of the laser scanning confocal microscope has not been utilized to capture the in-focus and three-dimensional cytoarchitecture of metal-impregnated cells. Here, we demonstrate how spectral confocal microscopy, typically reserved for fluorescent imaging, can be used to visualize metal-labeled tissues. This imaging does not involve the reflectance of metal particles, but rather the excitation of silver (or gold) nanoparticles and their putative surface plasmon resonance. To induce such resonance, silver or gold particles were excited with visible-wavelength laser lines (561 or 640 nm), and the maximal emission signal was collected at a shorter wavelength (i.e., higher energy state). Because the surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, our novel protocol holds enormous promise of a rebirth and further development of silver- and gold-based cell labeling protocols. DOI: http://dx.doi.org/10.7554/eLife.09388.001 PMID:26670545

Nondestructive 3D confocal laser imaging with deconvolution of seven whole stardust tracks with complementary XRF and quantitative analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greenberg, M.; Ebel, D.S.

2009-03-19

We present a nondestructive 3D system for analysis of whole Stardust tracks, using a combination of Laser Confocal Scanning Microscopy and synchrotron XRF. 3D deconvolution is used for optical corrections, and results of quantitative analyses of several tracks are presented. The Stardust mission to comet Wild 2 trapped many cometary and ISM particles in aerogel, leaving behind 'tracks' of melted silica aerogel on both sides of the collector. Collected particles and their tracks range in size from submicron to millimeter scale. Interstellar dust collected on the obverse of the aerogel collector is thought to have an average track length ofmore » {approx}15 {micro}m. It has been our goal to perform a total non-destructive 3D textural and XRF chemical analysis on both types of tracks. To that end, we use a combination of Laser Confocal Scanning Microscopy (LCSM) and X Ray Florescence (XRF) spectrometry. Utilized properly, the combination of 3D optical data and chemical data provides total nondestructive characterization of full tracks, prior to flattening or other destructive analysis methods. Our LCSM techniques allow imaging at 0.075 {micro}m/pixel, without the use of oil-based lenses. A full textural analysis on track No.82 is presented here as well as analysis of 6 additional tracks contained within 3 keystones (No.128, No.129 and No.140). We present a method of removing the axial distortion inherent in LCSM images, by means of a computational 3D Deconvolution algorithm, and present some preliminary experiments with computed point spread functions. The combination of 3D LCSM data and XRF data provides invaluable information, while preserving the integrity of the samples for further analysis. It is imperative that these samples, the first extraterrestrial solids returned since the Apollo era, be fully mapped nondestructively in 3D, to preserve the maximum amount of information prior to other, destructive analysis.« less

To boldly glow ... applications of laser scanning confocal microscopy in developmental biology.

PubMed

Paddock, S W

1994-05-01

The laser scanning confocal microscope (LSCM) is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.

Association of tongue brushing with the number of fungiform taste buds and taste perception: A preliminary study using confocal laser scanning microscopy in combination with a filter-paper disc method.

PubMed

Kobayashi, Junichi; Saito, Takehisa; Ito, Tetsufumi; Yoshimura, Hitoshi; Matsuda, Shinpei; Yoshida, Hisato; Fujita, Ryousuke; Sano, Kazuo

2017-12-01

The aim of this study was to investigate the association of tongue brushing with the number of fungiform taste buds and taste perception using a confocal laser scanning microscopy in combination with a filter-paper disc method (FPDM). Twenty-four subjects with or without a habit of tongue brushing (11 males and 13 females, 20-46 years old) participated in this study. Nine of the 24 subjects had no habit of tongue brushing (Group 1, n=9). Fifteen subjects had a habit of tongue brushing, and the brushing regions of the tongue were as follows: central region (Group 2, n=7), or entire region (Group 3, n=8) of the tongue dorsum. Using confocal laser scanning microscopy, the average number of taste buds per fungiform papilla (FP) was counted. Taste perception was evaluated using an FPDM. These observations were performed in the midlateral region of the tongue since the distribution of fungiform papillae is large in the midlateral region compared to that in the central region. The subjects in Group 3 showed a significantly decreased number of fungiform taste buds compared to Group 1 and Group 2. Group 3 also showed significantly higher FPDM scores than the other two groups. Excessive tongue brushing of the entire tongue dorsum, including the midlateral region, may have an association with the decreased number of FP and taste buds and decreased taste sensation. To avoid these conditions, instituting proper tongue brushing methods, such as limiting it to the central region of the tongue and using a light touch, is suggested and is important for the subjects who are eager to participate in tongue brushing. Copyright © 2017 Elsevier Ltd. All rights reserved.

Remineralization Potential of Three Different Dentifrices using Raman Spectroscopy and Confocal Laser Scanning Microscope.

PubMed

Job, Tisson V; Narayana, Girish T; Venkappa, Kishan K; Nathan, K Binu; Ahsan, Shameem; Harikaran, Jayakkodi

2018-04-01

Aim: The aim of this study was to compare the remineralization potential of three different dentifrices using Raman spectroscopy and confocal laser scanning microscopy (CLSM). Materials and methods: Totally, 30 extracted intact impacted third molar teeth were selected and the crown of each tooth in a group was separated from the root and longitudinally sectioned into four parts with each section under a subgroup, of which one section was an untreated section, the second and the third sections were demineralized in a demineralizing solution, and the third section was remineralized after demineralization. The teeth in the three groups were demineralized for 4 days and then treated with 0.21% sodium fluoride dentifrice with trical-cium phosphate, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and NovaMin for 14 days, following which the teeth surfaces were studied using Raman spec-troscopy and CLSM to assess the remineralization potential of the three dentifrices. The data were recorded and analyzed statistically. Results: Raman spectroscopic analysis revealed better remin-eralization with CPP-ACP, which was statistically significant from the groups treated with the NovaMin dentifrice and the fluoride-containing dentifrice.Confocal laser scanning microscopic examination also revealed significant differences between the three groups with the NovaMin-containing dentifrice demonstrating a greater remineralization of the surface when compared with the CPP-ACP dentifrice. The teeth samples treated with fluoride-containing dentifrice demonstrated the least reminer-alization among the three groups. Conclusion: It can be concluded that the demineralized samples of teeth treated with CPP-ACP showed the highest concentration of phosphate ions when analyzed using Raman spectroscopy, and the microscopic examination using confocal laser revealed a better surface remineralization of the demin-eralized samples when treated with the NovaMin technology. Clinical

Intensity calibration of a laser scanning confocal microscope based on concentrated dyes.

PubMed

Model, Michael A; Blank, James L

2006-10-01

To find water-soluble fluorescent dyes with absorption in various regions of the spectrum and investigate their utility as standards for laser scanning confocal microscopy. Several dyes were found to have characteristics required for fluorescence microscopy standards. The intensity of biological fluorescent specimens was measured against the emission of concentrated dyes. Results using different optics and different microscopes were compared. Slides based on concentrated dyes can be prepared in a highly reproducible manner and are stable under laser scanning. Normalized fluorescence of biological specimens remains consistent with different objective lenses and is tolerant to some mismatch in optical filters or imperfect pinhole alignment. Careful choice of scanning parameters is necessary to ensure linearity of intensity measurements. Concentrated dyes provide a robust and inexpensive intensity standard that can be used in basic research or clinical studies.

Carbon Dioxide Laser Ablation of Basal Cell Carcinoma with Visual Guidance by Reflectance Confocal Microscopy: A Proof of Principle Pilot Study

PubMed Central

Hibler, B.P.; Sierra, H.; Cordova, M.; Phillips, W.; Rajadhyaksha, M.; Nehal, K.S.; Rossi, A.M.

2016-01-01

Background Laser ablation is an alternative, non-surgical treatment modality for low-risk basal cell carcinoma (BCC); however, lack of confirmative tumour destruction or residual tumour presence has been a limiting factor to adoption. Reflectance confocal microscopy (RCM) provides non-invasive, cellular-level resolution imaging of the skin and is capable of identifying tumour. Objective To evaluate the use of RCM to guide carbon dioxide (CO2) laser ablation of BCC, confirm destruction, and correlate findings with histology. Methods RCM was used pre-ablation to evaluate for features of BCC. Ablation was performed with a CO2 laser, and the response rapidly assessed using handheld RCM to evaluate for residual tumour. Confirmative pathology was used to verify confocal imaging. Results RCM imaging identified tumour pre-ablation with features not identified on normal, surrounding skin. Post-ablation, RCM documented complete removal of tumour in six cases and residual tumour in two. Histologic examination identified the ablated area and confirmed clearance of tumour in the six aforementioned cases and corroborated confocal findings for residual tumour in the other two cases. Conclusions We report successful treatment of superficial and nodular BCC using CO2 laser ablation augmented by RCM imaging for pre-ablation guidance and verification of tumour removal post-ablation. Akin to complete circumferential and deep margin control techniques, using RCM helps to map peripheral and deep BCC margins to hone in on areas exhibiting persistent tumour after ablation. CO2 laser ablation visually guided by RCM can help circumvent previously cited limiting factors of laser ablation for tumour destruction by providing cellular-level resolution imaging of tumour and margin assessment in between each laser pass and post-ablation. PMID:26800657

Carbon dioxide laser ablation of basal cell carcinoma with visual guidance by reflectance confocal microscopy: a proof-of-principle pilot study.

PubMed

Hibler, B P; Sierra, H; Cordova, M; Phillips, W; Rajadhyaksha, M; Nehal, K S; Rossi, A M

2016-06-01

Laser ablation is an alternative, nonsurgical treatment modality for low-risk basal cell carcinoma (BCC). However, lack of confirmative tumour destruction or residual tumour presence has been a limiting factor to its adoption. Reflectance confocal microscopy (RCM) provides noninvasive, cellular-level resolution imaging of the skin and is capable of identifying tumour. To evaluate the use of RCM to guide carbon dioxide (CO2 ) laser ablation of BCC, confirm destruction and correlate findings with histology. RCM was used preablation to evaluate for features of BCC. Ablation was performed with a CO2 laser, and the response rapidly assessed using handheld RCM to evaluate for residual tumour. Confirmative pathology was used to verify confocal imaging. Preablation RCM imaging identified tumour with features not identified on normal, surrounding skin. Postablation, RCM documented complete removal of tumour in six cases and residual tumour in two. Histological examination identified the ablated area and confirmed clearance of tumour in the six aforementioned cases and corroborated confocal findings for residual tumour in the other two cases. We report successful treatment of superficial and nodular BCC using CO2 laser ablation augmented by RCM imaging for preablation guidance and verification of tumour removal postablation. Akin to complete circumferential and deep margin control techniques, using RCM helps to map peripheral and deep BCC margins to hone in on areas exhibiting persistent tumour after ablation. CO2 laser ablation visually guided by RCM can help circumvent previously cited limiting factors of laser ablation for tumour destruction by providing cellular-level resolution imaging of tumour and margin assessment in between each laser pass and postablation. © 2016 British Association of Dermatologists.

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).

Roughness of biopores and cracks in Bt-horizons by confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Leue, Martin; Gerke, Horst H.

2016-04-01

During preferential flow events in structured soils, the movement of water and reactive solutes is mostly restricted to larger inter-aggregate pores, cracks, and biopores. The micro-topography of such macropores in terms of pore shapes, geometry, and roughness is crucial for describing the exchange of water and solutes between macropores and the soil matrix. The objective of this study was to determine the surface roughness of intact structural surfaces from the Bt-horizon of Luvisols by confocal laser scanning microscopy. For this purpose, samples with the structural surface types including cracks with and without clay-organic coatings from Bt-horizons developed on loess and glacial till were compared. The surface roughness of these structures was calculated in terms of three parameters from selected surface regions of 0.36 mm² determined with a confocal laser scanning microscope of the type Keyence VK-X100K. These data were evaluated in terms of the root-mean-squared roughness, Rq, the curvature, Rku, and the ratio between surface area and base area, RA. Values of Rq and RA were smaller for coated as compared to uncoated cracks and earthworm burrows of the Bt-horizons from both parent materials. The results indicated that the illuviation of clayey material led to a "smoothing" of the crack surfaces, which was similar for the coarser textured till-Bt and the finer-textured loess-Bt surfaces. The roughness indicated by Rq and RA values was only slightly smaller and that indicated by Rku slightly higher for the structural surfaces from the loess as compared to those from the glacial till. These results suggest a minor importance of the parent material on the roughness of structural surfaces in the Bt-horizon. The similarity of Rq, RA, and Rku values between surfaces of earthworm burrows and uncoated cracks did not confirm an expected smoothing effect of the burrow walls by the earthworm. In contrast to burrow walls, root channels from the loess-Bt were smoother

In Vivo Confocal Intrinsic Optical Signal Identification of Localized Retinal Dysfunction

PubMed Central

Zhang, Qiu-Xiang; Lu, Rong-Wen; Curcio, Christine A.; Yao, Xin-Cheng

2012-01-01

Purpose. The purposes of this study were to investigate the physiological mechanism of stimulus-evoked fast intrinsic optical signals (IOSs) recorded in dynamic confocal imaging of the retina, and to demonstrate the feasibility of in vivo confocal IOS mapping of localized retinal dysfunctions. Methods. A rapid line-scan confocal ophthalmoscope was constructed to achieve in vivo confocal IOS imaging of frog (Rana pipiens) retinas at cellular resolution. In order to investigate the physiological mechanism of confocal IOS, comparative IOS and electroretinography (ERG) measurements were made using normal frog eyes activated by variable-intensity stimuli. A dynamic spatiotemporal filtering algorithm was developed to reject the contamination of hemodynamic changes on fast IOS recording. Laser-injured frog eyes were employed to test the potential of confocal IOS mapping of localized retinal dysfunctions. Results. Comparative IOS and ERG experiments revealed a close correlation between the confocal IOS and retinal ERG, particularly the ERG a-wave, which has been widely used to evaluate photoreceptor function. IOS imaging of laser-injured frog eyes indicated that the confocal IOS could unambiguously detect localized (30 μm) functional lesions in the retina before a morphological abnormality is detectable. Conclusions. The confocal IOS predominantly results from retinal photoreceptors, and can be used to map localized photoreceptor lesion in laser-injured frog eyes. We anticipate that confocal IOS imaging can provide applications in early detection of age-related macular degeneration, retinitis pigmentosa, and other retinal diseases that can cause pathological changes in the photoreceptors. PMID:23150616

Combination of synchrotron radiation-based Fourier transforms infrared microspectroscopy and confocal laser scanning microscopy to understand spatial heterogeneity in aquatic multispecies biofilms.

PubMed

Reuben, Sheela; Banas, Krzysztof; Banas, Agnieszka; Swarup, Sanjay

2014-11-01

Understanding the spatial heterogeneity within environmental biofilms can provide an insight into compartmentalization of different functions in biofilm communities. We used a non-destructive and label-free method by combining Synchrotron Radiation-based Fourier Transform Infrared Microspectroscopy (SR-FTIR) with Confocal Laser Scanning Microscopy (CLSM) to distinguish the spatial chemical changes within multispecies biofilms grown from natural storm waters in flow cells. Among the different surfaces tested for biofilm growth and optimal imaging, mylar membranes were most suited and it enabled successful spatial infrared imaging of natural biofilms for obtaining reliable and interpretable FTIR spectra. Time series analysis of biofilm growth showed that influx of water during biofilm growth, results in significant changes in biofilm formation. Early biofilms showed active nutrient acquisition and desiccation tolerance mechanisms corresponding with accumulation of secreted proteins. Statistical approach used for the evaluation of chemical spectra allowed for clustering and classification of various regions of the biofilm. Microheterogeneity was observed in the polymeric components of the biofilm matrix, including cellulose, glycocalyx and dextran-like molecules. Fructan and glycan-rich regions were distinguishable and glycocalyx was abundant in the strongly adhering peripheral regions of biofilms. Inner core showed coexistence of oxygen dimers and ferrihydrite that will likely support growth of Fe (II)-oxidising bacteria. The combined SR-FTIR microspectroscopy and CSLM approach for complex natural biofilms described here will be useful both in understanding heterogeneity of matrix components and in correlating functions of juxtaposed microbial species in complex natural biofilms with physicochemical microenvironment to which they are exposed. Copyright © 2014 Elsevier Ltd. All rights reserved.

UNC Pembroke Laser Scanning Confocal Microscopy Facility

DTIC Science & Technology

2016-04-29

cultures. INVASIVE FIRE ANTS Professor Lisa Kelly of UNC Pembroke has been trained on the new confocal system. Dr. Kelly’s research...interest in the trophic ecology of the invasive fire ant has begun to benefit from the wide field view and long working distances of a confocal imaging...of protein clearance pathways in living brain tissue cultures. INVASIVE FIRE ANTS Professor Lisa Kelly of UNC Pembroke has been trained on

Optical Design of Adaptive Optics Confocal Scanning Laser Ophthalmoscope with Two Deformable Mirrors.

PubMed

Yang, Jinsheng; Wang, Yuanyuan; Rao, Xuejun; Wei, Ling; Li, Xiqi; He, Yi

2017-01-01

We describe the optical design of a confocal scanning laser ophthalmoscope with two deformable mirrors. Spherical mirrors are used for pupil relay. Defocus aberration of the human eye is corrected by a Badal focusing structure and astigmatism aberration is corrected by a deformable mirror. The main optical system achieves a diffraction-limited performance through the entire scanning field (6 mm pupil, 3 degrees on pupil plane). The performance of the optical system, with correction of defocus and astigmatism, is also evaluated.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

PubMed

Gutierre, R C; Vannucci Campos, D; Mortara, R A; Coppi, A A; Arida, R M

2017-04-01

Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis. © 2017 Anatomical Society.

In vitro studies of Rickettsia-host cell interactions: Confocal laser scanning microscopy of Rickettsia helvetica-infected eukaryotic cell lines.

PubMed

Speck, Stephanie; Kern, Tanja; Aistleitner, Karin; Dilcher, Meik; Dobler, Gerhard; Essbauer, Sandra

2018-02-01

Rickettsia (R.) helvetica is the most prevalent rickettsia found in Ixodes ricinus ticks in Germany. Several studies reported antibodies against R. helvetica up to 12.5% in humans investigated, however, fulminant clinical cases are rare indicating a rather low pathogenicity compared to other rickettsiae. We investigated growth characteristics of R. helvetica isolate AS819 in two different eukaryotic cell lines with focus on ultra-structural changes of host cells during infection determined by confocal laser scanning microscopy. Further investigations included partially sequencing of rickA, sca4 and sca2 genes, which have been reported to encode proteins involved in cell-to-cell spread and virulence in some rickettsiae. R. helvetica grew constantly but slowly in both cell lines used. Confocal laser scanning microscopy revealed that the dissemination of R. helvetica AS819 in both cell lines was rather mediated by cell break-down and bacterial release than cell-to-cell spread. The cytoskeleton of both investigated eukaryotic cell lines was not altered. R. helvetica possesses rickA, but its expression is not sufficient to promote actin-based motility as demonstrated by confocal laser scanning microscopy. Hypothetical Sca2 and Sca4 proteins were deduced from nucleotide gene sequences but the predicted amino acid sequences were disrupted or truncated compared to other rickettsiae most likely resulting in non-functional proteins. Taken together, these results might give a first hint to the underlying causes of the reduced virulence and pathogenicity of R. helvetica.

Degeneration process of fungiform taste buds after severing the human chorda tympani nerve--observation by confocal laser scanning microscopy.

PubMed

Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Kato, Yuji; Manabe, Yasuhiro; Narita, Norihiko

2015-03-01

To elucidate the degeneration process of fungiform taste buds after severing the chorda tympani nerve (CTN) by confocal laser scanning microscopy in vivo. Prospective study. University hospital. Seven consecutive patients whose CTN was severed during tympanoplasty for middle ear cholesteatoma. Diagnostic. Preoperative and postoperative gustatory functions were assessed by electrogustometry (EGM). An average of 10 fungiform papillae (FP) in the midlateral region of the tongue were periodically observed, and the number of taste buds was counted using a confocal laser microscope. Among them, 2 to 3 reference FPs were selected based on the typical form of the FP or characteristic arrangements of taste pores. Observation was performed before surgery, 1 or 2 days after surgery, 2 or 3 times a week until 2 weeks after surgery, once a week between 2 and 4 weeks, and every 2 to 4 weeks thereafter until all taste buds had disappeared. EGM thresholds showed no response within 1 month after surgery in all patients. The initial change in the degeneration process was the disappearance of taste pores. The surface of taste buds became covered with epithelium. Finally, taste buds themselves atrofied and disappeared. The time course of degeneration differed depending upon individuals, each FP, and each taste bud. By employing the generalized linear mixed model under the Poisson distribution, it was calculated that all taste buds would disappear at around 50 days after surgery. Confocal laser scanning microscopy was useful for clarifying the degeneration process of fungiform taste buds.

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

Spatial organization of Pseudomonas aeruginosa biofilms probed by combined matrix-assisted laser desorption ionization mass spectrometry and confocal Raman microscopy.

PubMed

Masyuko, Rachel N; Lanni, Eric J; Driscoll, Callan M; Shrout, Joshua D; Sweedler, Jonathan V; Bohn, Paul W

2014-11-21

Bacteria growing as surface attached biofilms differ significantly from planktonic cells in several important traits that are reflected in the spatiotemporal organization of the cells and the extracellular polymeric substances they secrete. The structural and chemical features that define these biofilms are explored here using a combination of matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and confocal Raman microspectroscopies (CRM) to characterize and compare the composition and distribution of biomolecules found in biofilms and planktonic cells of the bacterium Pseudomonas aeruginosa. Three-day old P. aeruginosa biofilms show dramatic differences in molecular composition compared to planktonic cultures. CRM reveals that wild-type planktonic cell Raman spectra are characterized by bands linked to cellular constituents and are dominated by contributions from DNA- and RNA-related bands. In contrast, biofilm spectra are dominated by bands characteristic of glycolipids - rhamnolipids - polysaccharides and by secreted proteins. LDI MS was applied in turn to identify the rhamnolipids present in the biofilm. Experiments were also conducted using an acyl homoserine lactone quorum sensing-deficient mutant (ΔlasIΔrhlI), which is incapable of producing rhamnolipids. CRM and LDI MS analyses revealed that while molecular composition of the planktonic quorum sensing-deficient cells is similar to that of the wild-type planktonic cells, several compositional differences are observed in the mutant after biofilm growth, including complete absence of detectable rhamnolipids. CRM vibrational spectra of the mutant cells are very similar for planktonic and biofilm growth conditions, indicating that biofilm formation is greatly hindered in the absence of functioning quorum sensing machinery.

Attempt of correlative observation of morphological synaptic connectivity by combining confocal laser-scanning microscope and FIB-SEM for immunohistochemical staining technique.

PubMed

Sonomura, Takahiro; Furuta, Takahiro; Nakatani, Ikuko; Yamamoto, Yo; Honma, Satoru; Kaneko, Takeshi

2014-11-01

Ten years have passed since a serial block-face scanning electron microscopy (SBF-SEM) method was developed [1]. In this innovative method, samples were automatically sectioned with an ultramicrotome placed inside a scanning electron microscope column, and the block surfaces were imaged one after another by SEM to capture back-scattered electrons. The contrast-inverted images obtained by the SBF-SEM were very similar to those acquired using conventional TEM. SFB-SEM has made easy to acquire image stacks of the transmission electron microscopy (TEM) in the mesoscale, which is taken with the confocal laser-scanning microcopy(CF-LSM).Furthermore, serial-section SEM has been combined with the focused ion beam (FIB) milling method [2]. FIB-incorporated SEM (FIB-SEM) has enabled the acquisition of three-dimensional images with a higher z-axis resolution com- pared to ultramicrotome-equipped SEM.We tried immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in CF-LSM. Dendrites of neurons in the rat neostriatum were visualized using a recombinant viral vector. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively.We showed that conventional immuno-cytochemical staining for TEM was applicable to FIB-SEM. Furthermore, several synaptic contacts, which were thought to exist on the basis of CF-LSM findings, were confirmed with FIB-SEM, revealing the usefulness of the combined method of CF-LSM and FIB-SEM. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Epithelial innervation of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.

PubMed

Guthoff, Rudolf F; Wienss, Holger; Hahnel, Christian; Wree, Andreas

2005-07-01

Evaluation of a new method to visualize distribution and morphology of human corneal nerves (Adelta- and C-fibers) by means of fluorescence staining, confocal laser scanning microscopy, and 3-dimensional (3D) reconstruction. Trephinates of corneas with a diagnosis of Fuchs corneal dystrophy were sliced into layers of 200 microm thickness using a Draeger microkeratome (Storz, Germany). The anterior lamella was stained with the Life/Dead-Kit (Molecular Probes Inc.), examined by the confocal laser scanning microscope "Odyssey XL," step size between 0.5 and 1 microm, and optical sections were digitally 3D-reconstructed. Immediate staining of explanted corneas by the Life/Dead-Kit gave a complete picture of the nerves in the central human cornea. Thin nerves running parallel to the Bowman layer in the subepithelial plexus perforate the Bowman layer orthogonally through tube-like structures. Passing the Bowman layer, Adelta- and C-fibers can be clearly distinguished by fiber diameter, and, while running in the basal epithelial plexus, by their spatial arrangement. Adelta-fibers run straight and parallel to the Bowman layer underneath the basal cell layer. C-fibers, after a short run parallel to the Bowman layer, send off multiple branches penetrating epithelial cell layers orthogonally, ending blindly in invaginations of the superficial cells. In contrast to C-fibers, Adelta-fibers show characteristic bulbous formations when kinking into the basal epithelial plexus. Ex-vivo fluorescence staining of the cornea and 3D reconstructions of confocal scans provide a fast and easily reproducible tool to visualize nerves of the anterior living cornea at high resolution. This may help to clarify gross variations of nerve fiber patterns under various clinical and experimental conditions.

Re-description of Craspodema reflectans (Nematoda, Cyatholaimidae) using confocal laser scanning microscopy.

PubMed

Semprucci, Federica; Burattini, Sabrina

2015-06-12

Craspodema reflectans, erected by Gerlach 1964, is here re-described from some specimens recently found in the Maldivian archipelago and the implication of the new findings for the taxonomy of the Craspodema genus is discussed. Accordingly, an emended diagnosis of Craspodema genus and C. reflectans species are proposed. New data are also provided with the aid of the confocal laser scanning microscopy, using the natural fluorescence of the nematodes. The approach described here lays new foundations for the study of Museum collection material and it may be decisive for capture of new morphological details.

Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy

NASA Astrophysics Data System (ADS)

Clarke, David T.; Botchway, Stanley W.; Coles, Benjamin C.; Needham, Sarah R.; Roberts, Selene K.; Rolfe, Daniel J.; Tynan, Christopher J.; Ward, Andrew D.; Webb, Stephen E. D.; Yadav, Rahul; Zanetti-Domingues, Laura; Martin-Fernandez, Marisa L.

2011-09-01

Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena.

Dynamic Real-time Microscopy of the Urinary Tract Using Confocal Laser Endomicroscopy

PubMed Central

Wu, Katherine; Liu, Jen-Jane; Adams, Winifred; Sonn, Geoffrey A.; Mach, Kathleen E.; Pan, Ying; Beck, Andrew H.; Jensen, Kristin C.; Liao, Joseph C.

2014-01-01

OBJECTIVES To develop the diagnostic criteria for benign and neoplastic conditions of the urinary tract using probe-based confocal laser endomicroscopy (pCLE), a new technology for dynamic, in vivo imaging with micron-scale resolution. The suggested diagnostic criteria will formulate a guide for pCLE image interpretation in urology. METHODS Patients scheduled for transurethral resection of bladder tumor (TURBT) or nephrectomy were recruited. After white-light cystoscopy (WLC), fluorescein was administered as contrast. Different areas of the urinary tract were imaged with pCLE via direct contact between the confocal probe and the area of interest. Confocal images were subsequently compared with standard hematoxylin and eosin analysis. RESULTS pCLE images were collected from 66 participants, including 2 patients who underwent nephrectomy. We identified key features associated with different anatomic landmarks of the urinary tract, including the kidney, ureter, bladder, prostate, and urethra. In vivo pCLE of the bladder demonstrated distinct differences between normal mucosa and neoplastic tissue. Using mosaicing, a post hoc image-processing algorithm, individual image frames were juxtaposed to form wideangle views to better evaluate tissue microarchitecture. CONCLUSIONS In contrast to standard pathologic analysis of fixed tissue with hematoxylin and eosin, pCLE provides real time microscopy of the urinary tract to enable dynamic interrogation of benign and neoplastic tissues in vivo. The diagnostic criteria developed in this study will facilitate adaptation of pCLE for use in conjunction with WLC to expedite diagnosis of urinary tract pathology, particularly bladder cancer. PMID:21601243

Detection of fluorescent organic nanoparticles by confocal laser endomicroscopy in a rat model of Barrett's esophageal adenocarcinoma.

PubMed

Dassie, Elisa; Arcidiacono, Diletta; Wasiak, Iga; Damiano, Nunzio; Dall'Olmo, Luigi; Giacometti, Cinzia; Facchin, Sonia; Cassaro, Mauro; Guido, Ennio; De Lazzari, Franca; Marin, Oriano; Ciach, Tomasz; Fery-Forgues, Suzanne; Alberti, Alfredo; Battaglia, Giorgio; Realdon, Stefano

2015-01-01

For many years, novel strategies for cancer detection and treatment using nanoparticles (NPs) have been developed. Esophageal adenocarcinoma is the sixth leading cause of cancer-related deaths in Western countries, and despite recent advances in early detection and treatment, its prognosis is still very poor. This study investigated the use of fluorescent organic NPs as potential diagnostic tool in an experimental in vivo model of Barrett's esophageal adenocarcinoma. NPs were made of modified polysaccharides loaded with [4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran] (DCM), a well-known fluorescent dye. The NP periphery might or might not be decorated with ASYNYDA peptide that has an affinity for esophageal cancer cells. Non-operated and operated rats in which gastroesophageal reflux was surgically induced received both types of NPs (NP-DCM and NP-DCM-ASYNYDA) by intravenous route. Localization of mucosal NPs was assessed in vivo by confocal laser endomicroscopy, a technique which enables a "real time" and in situ visualization of the tissue at a cellular level. After injection of NP-DCM and NP-DCM-ASYNYDA, fluorescence was observed in rats affected by esophageal cancer, whereas no signal was observed in control non-operated rats, or in rats with simple esophagitis or Barrett's esophagus mucosa. Fluorescence was observable in vivo 30 minutes after the administration of NPs. Interestingly, NP-DCM-ASYNYDA induced strong fluorescence intensity 24 hours after administration. These observations suggested that NPs could reach the tumor cells, likely by enhanced permeability and retention effect, and the peptide ASYNYDA gave them high specificity for esophageal cancer cells. Thus, the combination of NP platform and confocal laser endomicroscopy could play an important role for highlighting esophageal cancer conditions. This result supports the potential of this strategy as a targeted carrier for photoactive and bioactive molecules in esophageal cancer

Near-Infrared Confocal Laser Reflectance Cytoarchitectural Imaging of the Substantia Nigra and Cerebellum in the Fresh Human Cadaver.

PubMed

Cheyuo, Cletus; Grand, Walter; Balos, Lucia L

2017-01-01

Cytoarchitectural neuroimaging remains critical for diagnosis of many brain diseases. Fluorescent dye-enhanced, near-infrared confocal in situ cellular imaging of the brain has been reported. However, impermeability of the blood-brain barrier to most fluorescent dyes limits clinical utility of this modality. The differential degree of reflectance from brain tissue with unenhanced near-infrared imaging may represent an alternative technique for in situ cytoarchitectural neuroimaging. We assessed the utility of unenhanced near-infrared confocal laser reflectance imaging of the cytoarchitecture of the cerebellum and substantia nigra in 2 fresh human cadaver brains using a confocal near-infrared laser probe. Cellular images based on near-infrared differential reflectance were captured at depths of 20-180 μm from the brain surface. Parts of the cerebellum and substantia nigra imaged using the probe were subsequently excised and stained with hematoxylin and eosin for histologic correlation. Near-infrared reflectance imaging revealed the 3-layered cytoarchitecture of the cerebellum, with Purkinje cells appearing hyperreflectant. In the substantia nigra, neurons appeared hyporeflectant with hyperreflectant neuromelanin cytoplasmic inclusions. Cytoarchitecture of the cerebellum and substantia nigra revealed on near-infrared imaging closely correlated with the histology on hematoxylin-eosin staining. We showed that unenhanced near-infrared reflectance imaging of fresh human cadaver brain can reliably identify and distinguish neurons and detailed cytoarchitecture of the cerebellum and substantia nigra. Copyright © 2016 Elsevier Inc. All rights reserved.

Confocal laser scanning microscopy and area-scale analysis used to quantify enamel surface textural changes from citric acid demineralization and salivary remineralization in vitro.

PubMed

Austin, R S; Giusca, C L; Macaulay, G; Moazzez, R; Bartlett, D W

2016-02-01

This paper investigates the application of confocal laser scanning microscopy to determine the effect of acid-mediated erosive enamel wear on the micro-texture of polished human enamel in vitro. Twenty polished enamel samples were prepared and subjected to a citric acid erosion and pooled human saliva remineralization model. Enamel surface microhardness was measured using a Knoop hardness tester, which confirmed that an early enamel erosion lesion was formed which was then subsequently completely remineralized. A confocal laser scanning microscope was used to capture high-resolution images of the enamel surfaces undergoing demineralization and remineralization. Area-scale analysis was used to identify the optimal feature size following which the surface texture was determined using the 3D (areal) texture parameter Sa. The Sa successfully characterized the enamel erosion and remineralization for the polished enamel samples (P<0.001). Areal surface texture characterization of the surface events occurring during enamel demineralization and remineralization requires optical imaging instrumentation with lateral resolution <2.5 μm, applied in combination with appropriate filtering in order to remove unwanted waviness and roughness. These techniques will facilitate the development of novel methods for measuring early enamel erosion lesions in natural enamel surfaces in vivo. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Sealing ability of three root-end filling materials prepared using an erbium: Yttrium aluminium garnet laser and endosonic tip evaluated by confocal laser scanning microscopy

PubMed Central

Nanjappa, A Salin; Ponnappa, KC; Nanjamma, KK; Ponappa, MC; Girish, Sabari; Nitin, Anita

2015-01-01

Aims: (1) To compare the sealing ability of mineral trioxide aggregate (MTA), Biodentine, and Chitra-calcium phosphate cement (CPC) when used as root-end filling, evaluated under confocal laser scanning microscope using Rhodamine B dye. (2) To evaluate effect of ultrasonic retroprep tip and an erbium:yttrium aluminium garnet (Er:YAG) laser on the integrity of three different root-end filling materials. Materials and Methods: The root canals of 80 extracted teeth were instrumented and obturated with gutta-percha. The apical 3 mm of each tooth was resected and 3 mm root-end preparation was made using ultrasonic tip (n = 30) and Er:YAG laser (n = 30). MTA, Biodentine, and Chitra-CPC were used to restore 10 teeth each. The samples were coated with varnish and after drying, they were immersed in Rhodamine B dye for 24 h. The teeth were then rinsed, sectioned longitudinally, and observed under confocal laser scanning microscope. Statistical Analysis Used: Data were analyzed using one-way analysis of variance (ANOVA) and a post-hoc Tukey's test at P < 0.05 (R software version 3.1.0). Results: Comparison of microleakage showed maximum peak value of 0.45 mm for Biodentine, 0.85 mm for MTA, and 1.05 mm for Chitra-CPC. The amount of dye penetration was found to be lesser in root ends prepared using Er:YAG laser when compared with ultrasonics, the difference was found to be statistically significant (P < 0.05). Conclusions: Root-end cavities prepared with Er:YAG laser and restored with Biodentine showed superior sealing ability compared to those prepared with ultrasonics. PMID:26180420

Acquisition of multiple image stacks with a confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Zuschratter, Werner; Steffen, Thomas; Braun, Katharina; Herzog, Andreas; Michaelis, Bernd; Scheich, Henning

1998-06-01

Image acquisition at high magnification is inevitably correlated with a limited view over the entire tissue section. To overcome this limitation we designed software for multiple image-stack acquisition (3D-MISA) in confocal laser scanning microscopy (CLSM). The system consists of a 4 channel Leica CLSM equipped with a high resolution z- scanning stage mounted on a xy-monitorized stage. The 3D- MISA software is implemented into the microscope scanning software and uses the microscope settings for the movements of the xy-stage. It allows storage and recall of 70 xyz- positions and the automatic 3D-scanning of image arrays between selected xyz-coordinates. The number of images within one array is limited only by the amount of disk space or memory available. Although for most applications the accuracy of the xy-scanning stage is sufficient for a precise alignment of tiled views, the software provides the possibility of an adjustable overlap between two image stacks by shifting the moving steps of the xy-scanning stage. After scanning a tiled image gallery of the extended focus-images of each channel will be displayed on a graphic monitor. In addition, a tiled image gallery of individual focal planes can be created. In summary, the 3D-MISA allows 3D-image acquisition of coherent regions in combination with high resolution of single images.

Emerging enhanced imaging technologies of the esophagus: spectroscopy, confocal laser endomicroscopy, and optical coherence tomography.

PubMed

Robles, Lourdes Y; Singh, Satish; Fisichella, Piero Marco

2015-05-15

Despite advances in diagnoses and therapy, esophageal adenocarcinoma remains a highly lethal neoplasm. Hence, a great interest has been placed in detecting early lesions and in the detection of Barrett esophagus (BE). Advanced imaging technologies of the esophagus have then been developed with the aim of improving biopsy sensitivity and detection of preplastic and neoplastic cells. The purpose of this article was to review emerging imaging technologies for esophageal pathology, spectroscopy, confocal laser endomicroscopy (CLE), and optical coherence tomography (OCT). We conducted a PubMed search using the search string "esophagus or esophageal or oesophageal or oesophagus" and "Barrett or esophageal neoplasm" and "spectroscopy or optical spectroscopy" and "confocal laser endomicroscopy" and "confocal microscopy" and "optical coherence tomography." The first and senior author separately reviewed all articles. Our search identified: 19 in vivo studies with spectroscopy that accounted for 1021 patients and 4 ex vivo studies; 14 clinical CLE in vivo studies that accounted for 941 patients and 1 ex vivo study with 13 patients; and 17 clinical OCT in vivo studies that accounted for 773 patients and 2 ex vivo studies. Human studies using spectroscopy had a very high sensitivity and specificity for the detection of BE. CLE showed a high interobserver agreement in diagnosing esophageal pathology and an accuracy of predicting neoplasia. We also found several clinical studies that reported excellent diagnostic sensitivity and specificity for the detection of BE using OCT. Advanced imaging technology for the detection of esophageal lesions is a promising field that aims to improve the detection of early esophageal lesions. Although advancing imaging techniques improve diagnostic sensitivities and specificities, their integration into diagnostic protocols has yet to be perfected. Copyright © 2015 Elsevier Inc. All rights reserved.

Observation of regenerated fungiform taste buds after severing the chorda tympani nerve using confocal laser scanning microscopy in vivo.

PubMed

Saito, Takehisa; Ito, Tetsufumi; Kato, Yuji; Yamada, Takechiyo; Manabe, Yasuhiro; Narita, Norihiko

2014-03-01

To evaluate whether regenerated fungiform taste buds after severing the chorda tympani nerve can be detected by confocal laser scanning microscopy in vivo. Retrospective study. University hospital. Six patients with a normal gustatory function (Group 1), 9 patients with taste function recovery after severing the CTN (Group 2), and 5 patients without taste function recovery (Group 3) were included. In Groups 2 and 3, canal wall up (closed) tympanoplasty or canal wall down with canal reconstruction tympanoplasty was performed in all patients. Diagnostic. The severed nerves were readapted or approximated on the temporalis muscle fascia used to reconstruct the eardrum during surgery. Preoperative and postoperative gustatory functions were assessed using electrogustometry. Twelve to 260 months after severing the CTN, the surface of the midlateral region of the tongue was observed with a confocal laser microscope. EGM thresholds showed no response 1 month after surgery in all patients of Groups 2 and 3. In Group 2, EGM thresholds showed recovery 1 to 2 years after surgery and before confocal microscopy (-1.3 ± 6.5 dB). There was a significant difference between Group 1 (-5.7 ± 2.0 dB; p < 0.01) and Group 2. In Group 3, EGM thresholds showed no response for more than 2 years. In the control group (Group 1), 0 to 16 taste buds were observed in each FP, and 55 (79.7%) of 69 FP contained at least 1 taste bud. The mean number of taste bud per papilla was 3.7 ± 3.6. In patients with a recovered taste function (Group 2), 0 to 8 taste buds were observed in each FP. In this group, 54 (56.2%) of 94 FP contained at least 1 taste bud. The mean number of taste bud per papilla was 2.0 ± 2.2 (p < 0.01). In Group 3, without recovery, the FP was atrophied, and no taste bud was observed. Regenerated fungiform taste bud could be observed in vivo using confocal laser scanning microscopy, indicating that regenerated taste bud can be detected without biopsy.

Adaptive optics for confocal laser scanning microscopy with adjustable pinhole

NASA Astrophysics Data System (ADS)

Yoo, Han Woong; van Royen, Martin E.; van Cappellen, Wiggert A.; Houtsmuller, Adriaan B.; Verhaegen, Michel; Schitter, Georg

2016-04-01

The pinhole plays an important role in confocal laser scanning microscopy (CLSM) for adaptive optics (AO) as well as in imaging, where the size of the pinhole denotes a trade-off between out-of-focus rejection and wavefront distortion. This contribution proposes an AO system for a commercial CLSM with an adjustable square pinhole to cope with such a trade-off. The proposed adjustable pinhole enables to calibrate the AO system and to evaluate the imaging performance. Experimental results with fluorescence beads on the coverslip and at a depth of 40 μm in the human hepatocellular carcinoma cell spheroid demonstrate that the proposed AO system can improve the image quality by the proposed calibration method. The proposed pinhole intensity ratio also indicates the image improvement by the AO correction in intensity as well as resolution.

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

NASA Astrophysics Data System (ADS)

Diaspro, Alberto; Chirico, Giberto; Federici, Federico; Cannone, Fabio; Beretta, Sabrina; Robello, Mauro; Olivini, Francesca; Ramoino, Paola

2001-07-01

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two- photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Confocal laser scanning microscopy of porcine skin: implications for human wound healing studies

PubMed Central

VARDAXIS, N. J.; BRANS, T. A.; BOON, M. E.; KREIS, R. W.; MARRES, L. M.

1997-01-01

The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model. PMID:9183682

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...

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.

[Significance of laser confocal tomography in diagnosis and monitoring of keratoconjunctivitis sicca].

PubMed

Safonova, T N; Gladkova, О V; Boev, V I

2016-01-01

Laser confocal tomography of the cornea enables studying ultrathin sections of corneal layers, which provides additional reliable information on tissue changes in keratoconjunctivitis sicca (KCS). To assess the significance of laser confocal tomography of the cornea in the diagnosis and monitoring of KCS. We investigated 38 eyes of 30 patients with severe KCS. The patients were divided into two groups. Group 1 (15 patients, 19 eyes) was prescribed cyclosporine А 0.05% instillations 2 times daily, artificial tears, and soft contact lenses. Group 2 (15 patients, 19 eyes) received only instillations of cyclosporine А 0.05% 2 times daily and artificial tears. Besides standard ophthalmic examination, additional tests were performed, namely Schirmer's test, tear break-up time test, fluorescein eye stain test, tear osmolarity test (TearLab System, USA), and Heidelberg retinal tomography of the cornea (HRT, Heidelberg Engineering GmbH, Germany). HRT findings revealed a 3 times shorter epithelization period and faster recovery of corneal transparency in group 1 as compared to group 2 (1.5 and 4.5 months, respectively). There was also an evident reduction in the number of immune cells in the cornea, most pronounced in group 1 at 3 months, which is indicative of inflammation termination. The use of HRT of the cornea in KCS patients allows real-time cellular level observation of corneal changes, which together with clinical findings and diagnostic tests not only confirms the diagnosis but also determines treatment effectiveness. It has been also found that soft contact lenses accelerate epithelization of the cornea and relieve inflammation of the ocular surface in KCS patients under cyclosporine A 0.05% instillation therapy. Transparency of financial activity: the authors have no financial interest in the submitted materials and methods.

Detection of fluorescent organic nanoparticles by confocal laser endomicroscopy in a rat model of Barrett’s esophageal adenocarcinoma

PubMed Central

Dassie, Elisa; Arcidiacono, Diletta; Wasiak, Iga; Damiano, Nunzio; Dall’Olmo, Luigi; Giacometti, Cinzia; Facchin, Sonia; Cassaro, Mauro; Guido, Ennio; De Lazzari, Franca; Marin, Oriano; Ciach, Tomasz; Fery-Forgues, Suzanne; Alberti, Alfredo; Battaglia, Giorgio; Realdon, Stefano

2015-01-01

For many years, novel strategies for cancer detection and treatment using nanoparticles (NPs) have been developed. Esophageal adenocarcinoma is the sixth leading cause of cancer-related deaths in Western countries, and despite recent advances in early detection and treatment, its prognosis is still very poor. This study investigated the use of fluorescent organic NPs as potential diagnostic tool in an experimental in vivo model of Barrett’s esophageal adenocarcinoma. NPs were made of modified polysaccharides loaded with [4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran] (DCM), a well-known fluorescent dye. The NP periphery might or might not be decorated with ASYNYDA peptide that has an affinity for esophageal cancer cells. Non-operated and operated rats in which gastroesophageal reflux was surgically induced received both types of NPs (NP-DCM and NP-DCM-ASYNYDA) by intravenous route. Localization of mucosal NPs was assessed in vivo by confocal laser endomicroscopy, a technique which enables a “real time” and in situ visualization of the tissue at a cellular level. After injection of NP-DCM and NP-DCM-ASYNYDA, fluorescence was observed in rats affected by esophageal cancer, whereas no signal was observed in control non-operated rats, or in rats with simple esophagitis or Barrett’s esophagus mucosa. Fluorescence was observable in vivo 30 minutes after the administration of NPs. Interestingly, NP-DCM-ASYNYDA induced strong fluorescence intensity 24 hours after administration. These observations suggested that NPs could reach the tumor cells, likely by enhanced permeability and retention effect, and the peptide ASYNYDA gave them high specificity for esophageal cancer cells. Thus, the combination of NP platform and confocal laser endomicroscopy could play an important role for highlighting esophageal cancer conditions. This result supports the potential of this strategy as a targeted carrier for photoactive and bioactive molecules in esophageal

Confocal Raman imaging of optical waveguides in LiNbO3 fabricated by ultrafast high-repetition rate laser-writing.

PubMed

Ródenas, Airán; Nejadmalayeri, Amir H; Jaque, Daniel; Herman, Peter

2008-09-01

We report on the confocal Raman characterization of the micro-structural lattice changes induced during the high-repetition rate ultrafast laser writing of buried optical waveguides in lithium niobate (LiNbO(3)) crystals. While the laser beam focal volume is characterized by a significant lattice expansion together with a high defect concentration, the adjacent waveguide zone is largely free of defects, undergoing only slight rearrangement of the oxygen octahedron in the LiNbO(3) lattice. The close proximity of these two zones has been found responsible for the propagation losses of the guided light. Subjacent laser-induced periodic micro-structures have been also observed inside the laser focal volume, and identified with a strong periodic distribution of lattice defects.

[Development and evaluation of a novel three-dimensional adjustable confocal laser-induced fluorescence detector].

PubMed

Liu, Fan; Yu, Shuxin; Tang, Tao; Sun, Yuanshe; Zhang, Weibing; Li, Tong

2011-09-01

Laser-induced fluorescence detector (LIFD) is one of the most sensitive detectors in analytical chemical files. Confocal optical configuration is widely used in LIFDs. Two effective approaches used to achieve the best signal to noise ratio (S/N) are increasing the confocal precision and minimizing the background noise. A novel three-dimensional adjustable confocal LIFD was developed, using a new three-dimensional adjustable supporter of reflector and modularized optical system. A detection limit (S/N = 3) of 1 x 10(-12) mol/L and a linear dynamic range of 3 orders of magnitude were obtained using fluorescein isothiocyanate (FITC) standard as the test sample. The noise level and drift levels were 8.0 x 10(-3) mV and 1.4 x 10(-3) mV/h, respectively, which were almost 10 times lower than before. And the stability of the LIFD was evaluated by five replicate injections of 5 x 10(-9) mol/L FITC, and the relative standard deviations (RSDs) of peak height and peak area were 0.38% and 0.41%, respectively. Further more, three biogenic amines, which were derivatized by FITC, were separated by high performance liquid chromatography (HPLC) and then detected by the novel LIFD. And the detection limits (S/N = 3) ranged 0.01 to 0.02 nmol/L, which were better than other methods. Therefore, the LIFD is highly sensitive, as well as shows a real low noise level and good reproducibility.

Development of confocal laser microscope system for examination of microscopic characteristics of radiophotoluminescence glass dosemeters.

PubMed

Maki, Daisuke; Ishii, Tetsuya; Sato, Fuminobu; Kato, Yushi; Yamamoto, Takayoshi; Iida, Toshiyuki

2011-03-01

A confocal laser microscope system was developed for the measurement of radiophotoluminescence (RPL) photons emitted from a minute alpha-ray-irradiated area in an RPL glass dosemeter. The system was composed mainly of an inverted-type microscope, an ultraviolet laser, an XY movable stage and photon-counting circuits. The photon-counting circuits were effective in the reduction of the background noise level in the measurement of RPL photons. The performance of this microscope system was examined by the observation of standard RPL glass samples irradiated using (241)Am alpha rays. The spatial resolution of this system was ∼ 3 μm, and with regard to the sensitivity of this system, a hit of more than four to five alpha rays in unit area produced enough amount of RPL photons to construct the image.

Signal and noise modeling in confocal laser scanning fluorescence microscopy.

PubMed

Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

2012-01-01

Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

Patent blue V and indocyanine green for fluorescence microimaging of human peritoneal carcinomatosis using probe-based confocal laser endomicroscopy.

PubMed

Abbaci, Muriel; Dartigues, Peggy; De Leeuw, Frederic; Soufan, Ranya; Fabre, Monique; Laplace-Builhé, Corinne

2016-12-01

Peritoneal carcinomatosis is a metastatic stage aggravating abdominal and pelvic cancer dissemination. The preoperative evaluation of lesions remains difficult today. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of tissue architecture and cellular details. This technology allows in vivo histological interpretation of tissue. The main limitation of pCLE for adoption in the clinic is the unavailability of fluorescent contrast agents. The aim of our study was to evaluate the staining performance of indocyanine green and patent blue V for histological diagnosis of pCLE images of pathological and non-pathological peritoneal tissue. We performed a correlative study with the histological gold standard on ex vivo human specimens from 25 patients operated for peritoneal carcinomatosis; 70 specimens were stained by topical application with ICG or patent blue V and then imaged with a probe-based confocal laser endomicroscope. A total of 350 pCLE images and 70 corresponding histological sections were randomly and blindly interpreted by two pathologists (PT1 and PT2). The images were first classified into two categories, tumoral versus non-tumoral, and a refined histological diagnosis was then given. All presented images were interpreted by PT1 (who received prior training on PCLE image reading) and PT2 (no training). 100 % sensitivity for PT1 and PT2 was noticed with tissues stained with ICG to differentiate tumoral and non-tumoral tissue. Global scores were always better for PT1 (major concordance between 86 and 94 %) than for PT2 (major concordance between 77 and 89 %) independently of the fluorescent dye when histological diagnosis was done on pCLE images. In conclusion, the pair ICG-pCLE offers the best combination for a non-trained pathologist for the interpretation of pCLE images from peritoneum.

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...

Preliminary identification of unicellular algal genus by using combined confocal resonance Raman spectroscopy with PCA and DPLS analysis

NASA Astrophysics Data System (ADS)

He, Shixuan; Xie, Wanyi; Zhang, Ping; Fang, Shaoxi; Li, Zhe; Tang, Peng; Gao, Xia; Guo, Jinsong; Tlili, Chaker; Wang, Deqiang

2018-02-01

The analysis of algae and dominant alga plays important roles in ecological and environmental fields since it can be used to forecast water bloom and control its potential deleterious effects. Herein, we combine in vivo confocal resonance Raman spectroscopy with multivariate analysis methods to preliminary identify the three algal genera in water blooms at unicellular scale. Statistical analysis of characteristic Raman peaks demonstrates that certain shifts and different normalized intensities, resulting from composition of different carotenoids, exist in Raman spectra of three algal cells. Principal component analysis (PCA) scores and corresponding loading weights show some differences from Raman spectral characteristics which are caused by vibrations of carotenoids in unicellular algae. Then, discriminant partial least squares (DPLS) classification method is used to verify the effectiveness of algal identification with confocal resonance Raman spectroscopy. Our results show that confocal resonance Raman spectroscopy combined with PCA and DPLS could handle the preliminary identification of dominant alga for forecasting and controlling of water blooms.

Penetration pattern of rhodamine dyes into enamel and dentin: confocal laser microscopy observation.

PubMed

Kwon, S R; Wertz, P W; Li, Y; Chan, D C N

2012-02-01

Enamel and dentin are susceptible to extrinsic and intrinsic stains. The purposes of this study were to determine the penetration pattern of Rhodamine B and dextran-conjugated Rhodamine B into the enamel and dentin as observed by confocal laser microscopy and to relate it to the penetration pattern of hydrogen peroxide commonly used as an active ingredient in tooth-whitening agents and high-molecular-weight staining molecules. Eighteen recently extracted human maxillary anterior teeth were used. Teeth were cleaned and painted with nail varnish except for the crown area above the cemento-enamel junction (CEJ). The painted teeth were then immersed in Rhodamine B and dextran-conjugated Rhodamine B (70 000 MW) for 4, 7, 10 and 15 days. Teeth were sliced to 3 mm thickness in transverse plane and mounted on a glass slide just prior to observation with confocal laser microscopy. Rhodamine B and dextran-conjugated Rhodamine B readily penetrated into the enamel and dentin when exposed for 4 and 7 days, respectively. Rhodamine B penetrated along the interprismatic spaces of the enamel into the dentin. The penetration was accentuated in sections with existing crack lines in the enamel. Rhodamine B was readily absorbed into the dentinal tubules at the dentino-enamel junction and continued to penetrate through the dentin via the dentinal tubules into the pre-dentin. Within the limitations of this study, it is concluded that Rhodamine B and dextran-conjugated Rhodamine B when applied to the external surface of the tooth readily penetrate into the enamel and dentin via the interprismatic spaces in the enamel and dentinal tubules in the dentin, suggesting that stain molecules and bleaching agents possibly exhibit similar penetration pathways. © 2011 The Authors. ICS © 2011 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Confocal endomicroscopy: Is it time to move on?

PubMed

Robles-Medranda, Carlos

2016-01-10

Confocal laser endomicroscopy permits in-vivo microscopy evaluation during endoscopy procedures. It can be used in all the parts of the gastrointestinal tract and includes: Esophagus, stomach, small bowel, colon, biliary tract through and endoscopic retrograde cholangiopancreatography and pancreas through needles during endoscopic ultrasound procedures. Many researches demonstrated a high correlation of results between confocal laser endomicroscopy and histopathology in the diagnosis of gastrointestinal lesions; with accuracy in about 86% to 96%. Moreover, in spite that histopathology remains the gold-standard technique for final diagnosis of any diseases; a considerable number of misdiagnosis rate could be present due to many factors such as interpretation mistakes, biopsy site inaccuracy, or number of biopsies. Theoretically; with the diagnostic accuracy rates of confocal laser endomicroscopy could help in a daily practice to improve diagnosis and treatment management of the patients. However, it is still not routinely used in the clinical practice due to many factors such as cost of the procedure, lack of codification and reimbursement in some countries, absence of standard of care indications, availability, physician image-interpretation training, medico-legal problems, and the role of the pathologist. These limitations are relative, and solutions could be found based on new researches focused to solve these barriers.

Laser scanning confocal microscopy and laser tweezers based experiments to understand dentine-bacteria interactions

NASA Astrophysics Data System (ADS)

Peng, Sum Chee; Mohanty, Samarendra; Gupta, P. K.; Kishen, Anil

2007-02-01

Failure of endodontic treatment is commonly due to Enterococcal infection. In this study influence of chemical treatments of type-I collagen membrane by chemical agents commonly used in endodontic treatment on Enterococcus faecalis cell adherence was evaluated. In order to determine the change in number of adhering bacteria after chemical treatment, confocal laser scanning microscopy was used. For this, overnight culture of E faecalis in All Culture broth was applied to chemically treated type-I collagen membrane. It was found that Ca(OH) II treated groups had statistically significant (p value=0.05) increase in population of bacteria adherence. The change in adhesion force between bacteria and collagen was determined by using optical tweezers (1064 nm). For this experiment, Type-I collagen membrane was soaked for 5 mins in a media that contained 50% all culture media and 50% saturated Ca(OH) II . The membrane was spread on the coverslip, on which diluted bacterial suspension was added. The force of laser tweezers on the bacteria was estimated at different trap power levels using viscous drag method and trapping stiffness was calculated using Equipartition theorem method. Presence of Ca(OH) II was found to increase the cell-substrate adherence force from 0.38pN to >2.1pN. Together, these experiments show that it was highly probable that the increase in adherence to collagen was due to a stronger adhesion in the presence of Ca (OH) II.

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 ...

Next generation of optical diagnostics for bladder cancer using probe-based confocal laser endomicroscopy

NASA Astrophysics Data System (ADS)

Liu, Jen-Jane; Chang, Timothy C.; Pan, Ying; Hsiao, Shelly T.; Mach, Kathleen E.; Jensen, Kristin C.; Liao, Joseph C.

2012-02-01

Real-time imaging with confocal laser endomicroscopy (CLE) probes that fit in standard endoscopes has emerged as a clinically feasible technology for optical biopsy of bladder cancer. Confocal images of normal, inflammatory, and neoplastic urothelium obtained with intravesical fluorescein can be differentiated by morphologic characteristics. We compiled a confocal atlas of the urinary tract using these diagnostic criteria to be used in a prospective diagnostic accuracy study. Patients scheduled to undergo transurethral resection of bladder tumor underwent white light cystoscopy (WLC), followed by CLE, and histologic confirmation of resected tissue. Areas that appeared normal by WLC were imaged and biopsied as controls. We imaged and prospectively analyzed 135 areas in 57 patients. We show that CLE improves the diagnostic accuracy of WLC for diagnosing benign tissue, low and high grade cancer. Interobserver studies showed a moderate level of agreement by urologists and nonclinical researchers. Despite morphologic differences between inflammation and cancer, real-time differentiation can still be challenging. Identification of bladder cancer-specific contrast agents could provide molecular specificity to CLE. By using fluorescently-labeled antibodies or peptides that bind to proteins expressed in bladder cancer, we have identified putative molecular contrast agents for targeted imaging with CLE. We describe one candidate agent - anti-CD47 - that was instilled into bladder specimens. The tumor and normal urothelium were imaged with CLE, with increased fluorescent signal demonstrated in areas of tumor compared to normal areas. Thus, cancer-specificity can be achieved using molecular contrast agents ex vivo in conjunction with CLE.

CCDiode: an optimal detector for laser confocal microscopes

NASA Astrophysics Data System (ADS)

Pawley, James B.; Blouke, Morley M.; Janesick, James R.

1996-04-01

The laser confocal microscope (LCM) is now an established research tool in biology and materials science. In biological applications, it is usually employed to detect the location of fluorescent market molecules and, under these conditions, signal levels from bright areas are often < 20 photons/pixel (from the specimen, assuming a standard 512 X 768, 1 sec. scan). Although this data rate limits the speed at which information can be derived from the specimen, saturation of the fluorophor, photobleaching of the dye, and phototoxicity prevent it being increased. Currently, most LCMs use photomultiplier tubes (PMT, QE equals 1 - 30% 400 - 900 nm). By contrast, rear-illuminated, scientific charge-coupled devices (CCD) now routinely readout the signal from square sensors approximately 30 micrometers on a side with a QE of 80 - 90%, a noise of only +/- 3 e-/pix and with no multiplicative noise. For this reason, in 1989, one of us (JJ) developed a rear-illuminated, single-channel Si sensor, called the Turbodiode, employing some of the sophisticated readout techniques used to measure charge in a scientific CCD. We are now extending this work to a device in which a single 36 X 36 micrometers sensor is read out through a low-noise FET charge amplifier with a reset circuit and then passed to a correlated, double-sampling digitizer. To maintain the desired +/- 3 e noise level at the relatively high data rate of 1 MHz, our new device utilizes 64 separate readout amplifier/digitizer systems, operating in sequence. The resulting detector is more compact, efficient and reliable than the PMT it replaces but as its sensitive area is smaller than that of a PMT, it will require auxiliary optics when used with any LCM having a large (mm) pinhole. As the signal light is parallel, a simple lens mounted axially and with the CCDiode at its focus would suffice. Future versions may use 3 X 3 or 5 X 5 arrays of sensors to `track' the confocal spot as it is deflected by inhomogeneities of the

Comparison of Fundus Autofluorescence Between Fundus Camera and Confocal Scanning Laser Ophthalmoscope–based Systems

PubMed Central

Park, Sung Pyo; Siringo, Frank S.; Pensec, Noelle; Hong, In Hwan; Sparrow, Janet; Barile, Gaetano; Tsang, Stephen H.; Chang, Stanley

2015-01-01

BACKGROUND AND OBJECTIVE To compare fundus autofluorescence (FAF) imaging via fundus camera (FC) and confocal scanning laser ophthalmoscope (cSLO). PATIENTS AND METHODS FAF images were obtained with a digital FC (530 to 580 nm excitation) and a cSLO (488 nm excitation). Two authors evaluated correlation of autofluorescence pattern, atrophic lesion size, and image quality between the two devices. RESULTS In 120 eyes, the autofluorescence pattern correlated in 86% of lesions. By lesion subtype, correlation rates were 100% in hemorrhage, 97% in geographic atrophy, 82% in flecks, 75% in drusen, 70% in exudates, 67% in pigment epithelial detachment, 50% in fibrous scars, and 33% in macular hole. The mean lesion size in geographic atrophy was 4.57 ± 2.3 mm2 via cSLO and 3.81 ± 1.94 mm2 via FC (P < .0001). Image quality favored cSLO in 71 eyes. CONCLUSION FAF images were highly correlated between the FC and cSLO. Differences between the two devices revealed contrasts. Multiple image capture and confocal optics yielded higher image contrast with the cSLO, although acquisition and exposure time was longer. PMID:24221461

Laser confocal microscope for analysis of 3013 inner container closure weld region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez-Rodriguez, M. J.

As part of the protocol to investigate the corrosion in the inner container closure weld region (ICCWR) a laser confocal microscope (LCM) was used to perform close visual examination of the surface and measurements of corrosion features on the surface. However, initial analysis of selected destructively evaluated (DE) containers using the LCM revealed several challenges for acquiring, processing and interpreting the data. These challenges include topography of the ICCWR sample, surface features, and the amount of surface area for collecting data at high magnification conditions. In FY17, the LCM parameters were investigated to identify the appropriate parameter values for datamore » acquisition and identification of regions of interest. Using these parameter values, selected DE containers were analyzed to determine the extent of the ICCWR to be examined.« less

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.

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.

Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

PubMed

Trepte, O; Rokahr, I; Andersson-Engels, S; Carlsson, K

1994-12-01

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Reflectance confocal microscopy-guided laser ablation of basal cell carcinomas: initial in vivo results

NASA Astrophysics Data System (ADS)

Sierra, Heidy; Cordova, Miguel; Yelamos, Oriol; Chen, Chih-Shan Jason; Rajadhyaksha, Milind

2017-02-01

Laser ablation offers a procedure for precise, fast and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided laser ablation approach offers cellular-level histopathology-like feedback directly on the patient, which may guide and help improve the efficacy of this procedure. We performed an initial study on 44 BCCs on 21 patients in vivo (based in an ex vivo bench-top study reported in our earlier papers), using a pulsed erbium: ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). Initial 10 lesions, the RCM imaging-guided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally, 34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, and the clearance of tumor is currently being monitored with follow-up imaging (i. e., no histopathology) at 3, 6 and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy in the wound, both immediately post-ablation and subsequently during recovery. The efficacy appears to be promising. However, further investigation and optimization to image over the entire wound (without missing any areas) need to be investigated.

Simultaneous measurement of refractive index and thickness by combining low-coherence interferometry and confocal optics.

PubMed

Kim, Seokhan; Na, Jihoon; Kim, Myoung Jin; Lee, Byeong Ha

2008-04-14

We propose and demonstrate novel methods that enable simultaneous measurements of the phase index, the group index, and the geometrical thickness of an optically transparent object by combining optical low-coherence interferometer and confocal optics. The low-coherence interferometer gives information relating the group index with the thickness, while the confocal optics allows access to the phase index related with the thickness of the sample. To relate these, two novel methods were devised. In the first method, the dispersion-induced broadening of the low-coherence envelop signal was utilized, and in the second method the frequency derivative of the phase index was directly obtained by taking the confocal measurements at several wavelengths. The measurements were made with eight different samples; B270, CaF2, two of BK7, two of fused silica, cover glass, and cigarette cover film. The average measurement errors of the first and the second methods were 0.123% and 0.061% in the geometrical thickness, 0.133% and 0.066% in the phase index, and 0.106% and 0.057% in the group index, respectively.

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.

The application of laser scanning confocal microscopy to the examination of hairs and textile fibers: an initial investigation.

PubMed

Kirkbride, K Paul; Tridico, Silvana R

2010-02-25

An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained. 2009 Elsevier Ireland Ltd. All rights reserved.

In vivo laser confocal microscopic analysis of corneal K-structures after keratorefractive surgery (LASIK and epi-LASIK).

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Tagawa, Kosaku; Sugiyama, Kazuhisa

2010-01-01

To demonstrate alterations of corneal K-structures (sub-Bowman's fibrous structures) after keratorefractive surgery by in vivo laser confocal microscopy and to look for association of K-structures with fluorescein-stained anterior corneal mosaic (ACM). Five patients (nine eyes) participated in this study. For four patients, one eye was evaluated after laser in situ keratomileusis (LASIK) and the other after epipolis-laser in situ keratomileusis (epi-LASIK). For one patient, the left eye was evaluated after epithelial debridement. A photograph of the ACM was obtained. Central corneal regions were scanned by Heidelberg Retina Tomograph 2 Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany). The ACM and K-structures disappeared in all corneas after epi-LASIK, but not after LASIK and epithelial debridement cornea. The presence of K-structures and ACM may be an index to identify eyes that had a previous refractive surgical procedure (surface ablation or LASIK) and be a health index of Bowman layer and adjacent anterior stroma. Copyright 2010, SLACK Incorporated.

Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy

NASA Astrophysics Data System (ADS)

Zhuo, S. M.; Chen, J. X.; Jiang, X. S.; Lu, K. C.; Xie, S. S.

2008-08-01

We combine reflectance confocal microscopy (RCM) with multiphoton microscopy (MPM) to image rat esophagus. The two imaging modalities allow detection of layered-resolved complementary information from esophagus. In the keratinizing layer, the keratinocytes boundaries can be characterized by RCM, while the keratinocytes cytoplasm (keratin) can be further imaged by multiphoton autofluorescence signal. In the epithelium, the epithelial cellular boundaries and nucleus can be detected by RCM, and MPM can be used for imaging epithelial cell cytoplasm and monitoring metabolic state of epithelium. In the stroma, multiphoton autofluorescence signal is used to image elastin and second harmonic generation signal is utilized to detect collagen, while RCM is used to determine the optical property of stroma. Overall, these results suggest that the combination of RCM and MPM has potential to provide more important and comprehensive information for early diagnosis of esophageal cancer.

Pupil engineering for a confocal reflectance line-scanning microscope

NASA Astrophysics Data System (ADS)

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

2011-03-01

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

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

Comparison of fungiform taste-bud distribution among age groups using confocal laser scanning microscopy in vivo in combination with gustatory function.

PubMed

Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Manabe, Yasuhiro; Sano, Kazuo

2016-04-01

The aim of this study was to compare the distribution of taste buds in fungiform papillae (FP) and gustatory function between young and elderly age groups. Confocal laser scanning microscopy was used because it allows many FP to be observed non-invasively in a short period of time. The age of participants (n = 211) varied from 20 to 83 yr. The tip and midlateral region of the tongue were observed. Taste buds in an average of 10 FP in each area were counted. A total of 2,350 FP at the tongue tip and 2,592 FP in the midlateral region could be observed. The average number of taste buds was similar among all age groups both at the tongue tip and in the midlateral region. The taste function, measured by electrogustometry, among participants 20-29 yr of age was significantly lower than that in the other age groups; however, there was no difference among any other age groups in taste function. These results indicate that the peripheral gustatory system is well maintained anatomically and functionally in elderly people. © 2016 Eur J Oral Sci.

Any Way You Slice It—A Comparison of Confocal Microscopy Techniques

PubMed Central

Jonkman, James

2015-01-01

The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research. PMID:25802490

Real time diagnosis of bladder cancer with probe-based confocal laser endomicroscopy

NASA Astrophysics Data System (ADS)

Liu, Jen-Jane; Wu, Katherine; Adams, Winifred; Hsiao, Shelly T.; Mach, Kathleen E.; Beck, Andrew H.; Jensen, Kristin C.; Liao, Joseph C.

2011-02-01

Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for in vivo optical imaging of the urinary tract. Particularly for bladder cancer, real time optical biopsy of suspected lesions will likely lead to improved management of bladder cancer. With pCLE, micron scale resolution is achieved with sterilizable imaging probes (1.4 or 2.6 mm diameter), which are compatible with standard cystoscopes and resectoscopes. Based on our initial experience to date (n = 66 patients), we have demonstrated the safety profile of intravesical fluorescein administration and established objective diagnostic criteria to differentiate between normal, benign, and neoplastic urothelium. Confocal images of normal bladder showed organized layers of umbrella cells, intermediate cells, and lamina propria. Low grade bladder cancer is characterized by densely packed monomorphic cells with central fibrovascular cores, whereas high grade cancer consists of highly disorganized microarchitecture and pleomorphic cells with indistinct cell borders. Currently, we are conducting a diagnostic accuracy study of pCLE for bladder cancer diagnosis. Patients scheduled to undergo transurethral resection of bladder tumor are recruited. Patients undergo first white light cystocopy (WLC), followed by pCLE, and finally histologic confirmation of the resected tissues. The diagnostic accuracy is determined both in real time by the operative surgeon and offline after additional image processing. Using histology as the standard, the sensitivity, specificity, positive and negative predictive value of WLC and WLC + pCLE are calculated. With additional validation, pCLE may prove to be a valuable adjunct to WLC for real time diagnosis of bladder cancer.

Optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy in retinal nerve fiber layer measurements of glaucoma patients.

PubMed

Fanihagh, Farsad; Kremmer, Stephan; Anastassiou, Gerasimos; Schallenberg, Maurice

2015-01-01

To determine the correlations and strength of association between different imaging systems in analyzing the retinal nerve fiber layer (RNFL) of glaucoma patients: optical coherence tomography (OCT), scanning laser polarimetry (SLP) and confocal scanning laser ophthalmoscopy (CSLO). 114 eyes of patients with moderate open angle glaucoma underwent spectral domain OCT (Topcon SD-OCT 2000 and Zeiss Cirrus HD-OCT), SLP (GDx VCC and GDx Pro) and CSLO (Heidelberg Retina Tomograph, HRT 3). Correlation coefficients were calculated between the structural parameters yielded by these examinations. The quantitative relationship between the measured RNFL thickness globally and for the four regions (superior, inferior, nasal, temporal) were evaluated with different regression models for all used imaging systems. The strongest correlation of RNFL measurements was found between devices using the same technology like GDx VCC and GDx Pro as well as Topcon OCT and Cirrus OCT. In glaucoma patients, the strongest associations (R²) were found between RNFL measurements of the two optical coherence tomography devices Topcon OCT and Cirrus OCT (R² = 0.513) and between GDx VCC and GDx Pro (R² = 0.451). The results of the OCTs and GDX Pro also had a strong quantitative relationship (Topcon OCT R² = 0.339 and Cirrus OCT R² = 0.347). GDx VCC and the OCTs showed a mild to moderate association (Topcon OCT R² = 0.207 and Cirrus OCT R² = 0.258). The confocal scanning laser ophthalmoscopy (HRT 3) had the lowest association to all other devices (Topcon OCT R² = 0.254, Cirrus OCT R² = 0.158, GDx Pro R² = 0.086 and GDx VCC R² = 0.1). The measurements of the RNFL in glaucoma patients reveal a high correlation of OCT and GDx devices because OCTs can measure all major retinal layers and SLP can detect nerve fibers allowing a comparison between the results of this devices. However, CSLO by means of HRT topography can only measure height values of the retinal surface but it cannot distinguish

Application of laser scanning confocal microscopy in the soft tissue exquisite structure for 3D scan

PubMed Central

Zhang, Zhaoqiang; Ibrahim, Mohamed; Fu, Yang; Wu, Xujia; Ren, Fei; Chen, Lei

2018-01-01

Three-dimensional (3D) printing is a new developing technology for printing individualized materials swiftly and precisely in the field of biological medicine (especially tissue-engineered materials). Prior to printing, it is necessary to scan the structure of the natural biological tissue, then construct the 3D printing digital model through optimizing the scanned data. By searching the literatures, magazines at home and abroad, this article reviewed the current status, main processes and matters needing attention of confocal laser scanning microscope (LSCM) in the application of soft tissue fine structure 3D scanning, empathizing the significance of LSCM in this field. PMID:29755838

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Design of small confocal endo-microscopic probe working under multiwavelength environment

NASA Astrophysics Data System (ADS)

Kim, Young-Duk; Ahn, MyoungKi; Gweon, Dae-Gab

2010-02-01

Recently, optical imaging system is widely used in medical purpose. By using optical imaging system specific diseases can be easily diagnosed at early stage because optical imaging system has high resolution performance and various imaging method. These methods are used to get high resolution image of human body and can be used to verify whether the cell is infected by virus. Confocal microscope is one of the famous imaging systems which is used for in-vivo imaging. Because most of diseases are accompanied with cellular level changes, doctors can diagnosis at early stage by observing the cellular image of human organ. Current research is focused in the development of endo-microscope that has great advantage in accessibility to human body. In this research, I designed small probe that is connected to confocal microscope through optical fiber bundle and work as endo-microscope. And this small probe is mainly designed to correct chromatic aberration to use various laser sources for both fluorescence type and reflection type confocal images. By using two kinds of laser sources at the same time we demonstrated multi-modality confocal endo-microscope.

Programmable Illumination and High-Speed, Multi-Wavelength, Confocal Microscopy Using a Digital Micromirror

PubMed Central

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

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

Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts

PubMed Central

Ozbay, Baris N.; Losacco, Justin T.; Cormack, Robert; Weir, Richard; Bright, Victor M.; Gopinath, Juliet T.; Restrepo, Diego; Gibson, Emily A.

2015-01-01

We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2 g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ~12 µm and an axial scan range of ~80 µm. The lateral field-of-view is 300 µm, and the lateral resolution is 1.8 µm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP). PMID:26030555

Chlorophyll and its degradation products in the two-spotted spider mite, Tetranychus urticae: observations using epifluorescence and confocal laser scanning microscopy.

PubMed

Occhipinti, Andrea; Maffei, Massimo E

2013-10-01

Chlorophyll and chlorophyll degradation products were observed in the two-spotted spider mite (Tetranychus urticae) using epifluorescence microscopy (EFM) and confocal laser scanning microscopy (CLSM). A clear red fluorescence (EFM) and a fluorescence induced by a laser wavelength of 650 nm (CLSM) were observed. In the lateral caeca, in the ventriculus and in the excretory organ, a bright light blue fluorescence was observed in close association with chlorophyll by using EFM. The same material can be localized with CLSM by using a laser with a wavelength of 488 nm. By comparison with synthetic guanine, this bright fluorescence is supposed to be guanine. The presence of guanine fluorescence in the mite pellets confirms this hypothesis. A possible mechanism for guanine formation is discussed.

Morphological characteristics of the optic nerve evaluated by confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population from the city of Barcelona.

PubMed

Fallon, M; Pazos, M; Morilla, A; Sebastián, M A; Xancó, R; Mora, C; Calderón, B; Vega, Z; Antón, A

2015-11-01

To evaluate morphological parameters of optic disc and retinal nerve fiber layer (RNFL) examined with confocal laser tomography (HRT3) and laser polarimetry (GDx-VCC) in a normal population, and analyze correlations of these parameters with demographic variables. Cross-sectional study in the context of a glaucoma screening campaign in the primary care center of Barcelona. The individuals selected were non-hypertensive Mediterranean Caucasians with risk for glaucoma development (individuals≥60 years old or≥40 years old with family history of glaucoma or intraocular pressure or myopia>3diopter). All subjects underwent a complete ophthalmic examination, confocal laser tomography (HRT3) and scanning laser polarimetry (GDX-VCC), subjects with results within normal limits only being included. Structural parameters were analyzed along with age, refraction, and pachymetry based on the Spearman rank correlation test. A total of 224 subjects included, with a mean age of 63.4±11.1 years. Disc areas, excavation and ring area were 2.14±0.52mm(2), 0.44±0.34mm (2) and 1.69±0.38mm(2), respectively. The mean RNFL (GDX) was 55.9±6.9μm. Age was correlated with lower ring volume, highest rate of cup shape measure, largest mean and maximum cup depth, lower nerve fiber index (NFI) and RNFL (all p-values below .05). The mean values and distribution of several parameters of the papilla and the RNFL in normal Mediterranean Caucasians population are presented. A loss of thickness of the RNFL, ring thinning, and enlarged cup was observed with increased age. Copyright © 2014 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

NASA Astrophysics Data System (ADS)

Meakin, J. P.; Speight, J. D.; Sheridan, R. S.; Bradshaw, A.; Harris, I. R.; Williams, A. J.; Walton, A.

2016-08-01

Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

Interference Confocal Microscope Integrated with Spatial Phase Shifter.

PubMed

Wang, Weibo; Gu, Kang; You, Xiaoyu; Tan, Jiubin; Liu, Jian

2016-08-24

We present an interference confocal microscope (ICM) with a new single-body four-step simultaneous phase-shifter device designed to obtain high immunity to vibration. The proposed ICM combines the respective advantages of simultaneous phase shifting interferometry and bipolar differential confocal microscopy to obtain high axis resolution, large dynamic range, and reduce the sensitivity to vibration and reflectance disturbance seamlessly. A compact single body spatial phase shifter is added to capture four phase-shifted interference signals simultaneously without time delay and construct a stable and space-saving simplified interference confocal microscope system. The test result can be obtained by combining the interference phase response and the bipolar property of differential confocal microscopy without phase unwrapping. Experiments prove that the proposed microscope is capable of providing stable measurements with 1 nm of axial depth resolution for either low- or high-numerical aperture objective lenses.

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.

Multicolor probe-based confocal laser endomicroscopy: a new world for in vivo and real-time cellular imaging

NASA Astrophysics Data System (ADS)

Vercauteren, Tom; Doussoux, François; Cazaux, Matthieu; Schmid, Guillaume; Linard, Nicolas; Durin, Marie-Amélie; Gharbi, Hédi; Lacombe, François

2013-03-01

Since its inception in the field of in vivo imaging, endomicroscopy through optical fiber bundles, or probe-based Confocal Laser Endomicroscopy (pCLE), has extensively proven the benefit of in situ and real-time examination of living tissues at the microscopic scale. By continuously increasing image quality, reducing invasiveness and improving system ergonomics, Mauna Kea Technologies has turned pCLE not only into an irreplaceable research instrument for small animal imaging, but also into an accurate clinical decision making tool with applications as diverse as gastrointestinal endoscopy, pulmonology and urology. The current implementation of pCLE relies on a single fluorescence spectral band making different sources of in vivo information challenging to distinguish. Extending the pCLE approach to multi-color endomicroscopy therefore appears as a natural plan. Coupling simultaneous multi-laser excitation with minimally invasive, microscopic resolution, thin and flexible optics, allows the fusion of complementary and valuable biological information, thus paving the way to a combination of morphological and functional imaging. This paper will detail the architecture of a new system, Cellvizio Dual Band, capable of video rate in vivo and in situ multi-spectral fluorescence imaging with a microscopic resolution. In its standard configuration, the system simultaneously operates at 488 and 660 nm, where it automatically performs the necessary spectral, photometric and geometric calibrations to provide unambiguously co-registered images in real-time. The main hardware and software features, including calibration procedures and sub-micron registration algorithms, will be presented as well as a panorama of its current applications, illustrated with recent results in the field of pre-clinical imaging.

Laser excited confocal microscope fluorescence scanner and method

DOEpatents

Mathies, Richard A.; Peck, Konan

1992-01-01

A fluorescent scanner for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier including a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from said volume to provide a display of the separated sample.

Spatiotemporal closure of fractional laser-ablated channels imaged by optical coherence tomography and reflectance confocal microscopy.

PubMed

Banzhaf, Christina A; Wind, Bas S; Mogensen, Mette; Meesters, Arne A; Paasch, Uwe; Wolkerstorfer, Albert; Haedersdal, Merete

2016-02-01

Optical coherence tomography (OCT) and reflectance confocal microscopy (RCM) offer high-resolution optical imaging of the skin, which may provide benefit in the context of laser-assisted drug delivery. We aimed to characterize postoperative healing of ablative fractional laser (AFXL)-induced channels and dynamics in their spatiotemporal closure using in vivo OCT and RCM techniques. The inner forearm of healthy subjects (n = 6) was exposed to 10,600 nm fractional CO2 laser using 5 and 25% densities, 120 μm beam diameter, 5, 15, and 25 mJ/microbeam. Treatment sites were scanned with OCT to evaluate closure of AFXL-channels and RCM to evaluate subsequent re-epithelialization. OCT and RCM identified laser channels in epidermis and upper dermis as black, ablated tissue defects surrounded by characteristic hyper-and hyporeflective zones. OCT imaged individual laser channels of the entire laser grid, and RCM imaged epidermal cellular and structural changes around a single laser channel to the depth of the dermoepidermal junction (DEJ) and upper papillary dermis. OCT images visualized a heterogeneous material in the lower part of open laser channels, indicating tissue fluid. By OCT the median percentage of open channels was evaluated at several time points within the first 24 hours and laser channels were found to gradually close, depending on the used energy level. Thus, at 5 mJ/microbeam, 87% (range 73-100%) of channels were open one hour after laser exposure, which declined to 27% (range 20-100%) and 20% (range 7-93%) at 12 and 24 hours after laser exposure, respectively. At 25 mJ/microbeam, 100% (range 100-100%) of channels were open 1 hour after laser exposure while 53% (range 33-100%) and 40% (range 0-100%) remained open at 12 and 24 hours after exposure. Median depth and width of open channels decreased over time depending of applied energy. RCM verified initial re-epithelialization from day 2 for all energy levels used. Morphology of ablation defects by OCT and

Lateral resolution testing of a novel developed confocal microscopic imaging system

NASA Astrophysics Data System (ADS)

Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

2015-10-01

Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.

Upconversion fiber-optic confocal microscopy under near-infrared pumping.

PubMed

Kim, Do-Hyun; Kang, Jin U; Ilev, Ilko K

2008-03-01

We present a simple upconversion fiber-optic confocal microscope design using a near-infrared laser for pumping of a rare-earth-doped glass powder. The nonlinear optical frequency conversion process is highly efficient with more than 2% upconversion fluorescence efficiency at a near-infrared pumping wavelength of 1.55 microm. The upconversion confocal design allows the use of conventional Si detectors and 1.55 microm near-infrared pump light. The lateral and axial resolutions of the system were equal to or better than 1.10 and 13.11 microm, respectively.

3D Volumetric Analysis of Fluid Inclusions Using Confocal Microscopy

NASA Astrophysics Data System (ADS)

Proussevitch, A.; Mulukutla, G.; Sahagian, D.; Bodnar, B.

2009-05-01

Fluid inclusions preserve valuable information regarding hydrothermal, metamorphic, and magmatic processes. The molar quantities of liquid and gaseous components in the inclusions can be estimated from their volumetric measurements at room temperatures combined with knowledge of the PVTX properties of the fluid and homogenization temperatures. Thus, accurate measurements of inclusion volumes and their two phase components are critical. One of the greatest advantages of the Laser Scanning Confocal Microscopy (LSCM) in application to fluid inclsion analsyis is that it is affordable for large numbers of samples, given the appropriate software analysis tools and methodology. Our present work is directed toward developing those tools and methods. For the last decade LSCM has been considered as a potential method for inclusion volume measurements. Nevertheless, the adequate and accurate measurement by LSCM has not yet been successful for fluid inclusions containing non-fluorescing fluids due to many technical challenges in image analysis despite the fact that the cost of collecting raw LSCM imagery has dramatically decreased in recent years. These problems mostly relate to image analysis methodology and software tools that are needed for pre-processing and image segmentation, which enable solid, liquid and gaseous components to be delineated. Other challenges involve image quality and contrast, which is controlled by fluorescence of the material (most aqueous fluid inclusions do not fluoresce at the appropriate laser wavelengths), material optical properties, and application of transmitted and/or reflected confocal illumination. In this work we have identified the key problems of image analysis and propose some potential solutions. For instance, we found that better contrast of pseudo-confocal transmitted light images could be overlayed with poor-contrast true-confocal reflected light images within the same stack of z-ordered slices. This approach allows one to narrow

Use of portable devices and confocal Raman spectrometers at different wavelength to obtain the spectral information of the main organic components in tomato (Solanum lycopersicum) fruits

NASA Astrophysics Data System (ADS)

Trebolazabala, Josu; Maguregui, Maite; Morillas, Héctor; de Diego, Alberto; Madariaga, Juan Manuel

2013-03-01

Tomato (Solanum lycopersicum) fruit samples, in two ripening stages, ripe (red) and unripe (green), collected from a cultivar in the North of Spain (Barrika, Basque Country), were analyzed directly, without any sample pretreatment, with two different Raman instruments (portable spectrometer coupled to a micro-videocamera and a confocal Raman microscope), using two different laser excitation wavelengths (514 and 785 nm, only for the confocal microscope). The combined use of these laser excitation wavelengths allows obtaining, in a short period of time, the maximum spectral information about the main organic compounds present in this fruit. The major identified components of unripe tomatoes were cutin and cuticular waxes. On the other hand, the main components on ripe tomatoes were carotenes, polyphenoles and polysaccharides. Among the carotenes, it was possible to distinguish the presence of lycopene from β-carotene with the help of both excitation wavelengths, but specially using the 514 nm one, which revealed specific overtones and combination tones of this type of carotene.

Confocal microscopy imaging of the biofilm matrix.

PubMed

Schlafer, Sebastian; Meyer, Rikke L

2017-07-01

The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens. Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine the concentration of solutes and the diffusive properties of the biofilm matrix. Copyright © 2016 Elsevier B.V. All rights reserved.

Laser excited confocal microscope fluorescence scanner and method

DOEpatents

Mathies, R.A.; Peck, K.

1992-02-25

A fluorescent scanner is designed for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier. The scanner includes a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from the volume to provide a display of the separated sample. 8 figs.

Spinning-disk confocal microscopy: present technology and future trends.

PubMed

Oreopoulos, John; Berman, Richard; Browne, Mark

2014-01-01

Live-cell imaging requires not only high temporal resolution but also illumination powers low enough to minimize photodamage. Traditional single-point laser scanning confocal microscopy (LSCM) is generally limited by both the relatively slow speed at which it can acquire optical sections by serial raster scanning (a few Hz) and the higher potential for phototoxicity. These limitations have driven the development of rapid, parallel forms of confocal microscopy, the most popular of which is the spinning-disk confocal microscope (SDCM). Here, we briefly introduce the SDCM technique, discuss its strengths and weaknesses against LSCM, and update the reader on some recent developments in SDCM technology that improve its performance and expand its utility for life science research now and in the future. © 2014 Elsevier Inc. All rights reserved.

Confocal laser scanning microscopy detection of chlorophylls and carotenoids in chloroplasts and chromoplasts of tomato fruit.

PubMed

D'Andrea, Lucio; Amenós, Montse; Rodríguez-Concepción, Manuel

2014-01-01

Plant cells are unique among eukaryotic cells because of the presence of plastids, including chloroplasts and chromoplasts. Chloroplasts are found in green tissues and harbor the photosynthetic machinery (including chlorophyll molecules), while chromoplasts are present in non-photosynthetic tissues and accumulate large amounts of carotenoids. During tomato fruit development, chloroplasts are converted into chromoplasts that accumulate high levels of lycopene, a linear carotenoid responsible for the characteristic red color of ripe fruit. Here, we describe a simple and fast method to detect both types of fully differentiated plastids (chloroplasts and chromoplasts), as well as intermediate stages, in fresh tomato fruits. The method is based on the differential autofluorescence of chlorophylls and carotenoids (lycopene) detected by Confocal Laser Scanning Microscopy.

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Lemelle, A.; Veksler, B.; Kozhevnikov, I. S.; Akchurin, G. G.; Piletsky, S. A.; Meglinski, I.

2009-01-01

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres.

Use of digital micromirror devices as dynamic pinhole arrays for adaptive confocal fluorescence microscopy

NASA Astrophysics Data System (ADS)

Pozzi, Paolo; Wilding, Dean; Soloviev, Oleg; Vdovin, Gleb; Verhaegen, Michel

2018-02-01

In this work, we present a new confocal laser scanning microscope capable to perform sensorless wavefront optimization in real time. The device is a parallelized laser scanning microscope in which the excitation light is structured in a lattice of spots by a spatial light modulator, while a deformable mirror provides aberration correction and scanning. A binary DMD is positioned in an image plane of the detection optical path, acting as a dynamic array of reflective confocal pinholes, images by a high performance cmos camera. A second camera detects images of the light rejected by the pinholes for sensorless aberration correction.

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.

Raman beam combining for laser brightness enhancement

DOEpatents

Dawson, Jay W.; Allen, Graham S.; Pax, Paul H.; Heebner, John E.; Sridharan, Arun K.; Rubenchik, Alexander M.; Barty, Chrisopher B. J.

2015-10-27

An optical source capable of enhanced scaling of pulse energy and brightness utilizes an ensemble of single-aperture fiber lasers as pump sources, with each such fiber laser operating at acceptable pulse energy levels. Beam combining involves stimulated Raman scattering using a Stokes' shifted seed beam, the latter of which is optimized in terms of its temporal and spectral properties. Beams from fiber lasers can thus be combined to attain pulses with peak energies in excess of the fiber laser self-focusing limit of 4 MW while retaining the advantages of a fiber laser system of high average power with good beam quality.

In-situ observation of recrystallization in an AlMgScZr alloy using confocal laser scanning microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taendl, J., E-mail: johannes.taendl@tugraz.atl; Nambu, S.; Orthacker, A.

2015-10-15

In this work we present a novel in-situ approach to study the recrystallization behavior of age hardening alloys. We use confocal laser scanning microscopy (CLSM) at 400 °C to investigate the static recrystallization of an AlMg4Sc0.4Zr0.12 alloy in-situ. The results are combined with electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) analyses. It was found that CLSM is a powerful tool to visualize both the local initiation and temporal sequence of recrystallization. After fast nucleation and initial growth, the grain growth rate decreases and the grain boundary migration stops after some minutes due to Zener pinning from Al{sub 3}(Sc,Zr)more » precipitates produced during the heat treatment. EBSD and TEM analyses confirm both the boundary movements and the particle-boundary interactions. - Highlights: • First time that CLSM is used to study recrystallization in-situ. • The start and end of recrystallization can be directly observed. • The procedure is easy to apply and requires only simple data interpretation. • In-situ observations on the surface correlate to modifications inside the bulk. • In-situ observations correlate to EBSD and EFTEM analyses.« less

The noninvasive retro-mode imaging of confocal scanning laser ophthalmoscopy in myopic maculopathy: a prospective observational study

PubMed Central

Su, Y; Zhang, X; Wu, K; Ji, Y; Zuo, C; Li, M; Wen, F

2014-01-01

Purpose To investigate the morphological features of myopic maculopathy with a new and noninvasive retro-mode imaging (RMI) technique using a confocal scanning laser ophthalmoscope. Methods A total of 42 patients (69 eyes) with myopic maculopathy were included. RMI combined with fundus photography, fundus fluorescein angiography, and optical coherence tomography together were used to observe and evaluate the morphological features of disease. Results Four in 4 eyes (100%) with macular retinoschisis were found with a characteristic pattern by RMI (firework pattern centrally with surrounding fingerprint pattern). Twenty-four in 24 eyes (100%) with pigment proliferation were found by RMI as dark plain patches, and 23 in 24 eyes with hemorrhage (95.8%) were found by RMI as gray bump. Atrophy of different degrees (12 in 14 eyes, 85.7%) was found by RMI as an area of pseudo-3D choroidal vessels or a fuzzy shadow but both without a clear boundary. Choroidal neovascularization (12 in 16 eyes, 75%) was identified laboriously by RMI as a vague raised region. Lacquer cracks were difficult to figure out in RMI. Conclusions Retinoschisis, pigment proliferation, hemorrhage, and atrophy secondary to myopic maculopathy have characteristic morphologic features in RMI; however, choroidal neovascularization and lacquer crack are not easily distinguishable in RMI. PMID:24924440

Simulation with Python on transverse modes of the symmetric confocal resonator

NASA Astrophysics Data System (ADS)

Wang, Qing Hua; Qi, Jing; Ji, Yun Jing; Song, Yang; Li, Zhenhua

2017-08-01

Python is a popular open-source programming language that can be used to simulate various optical phenomena. We have developed a suite of programs to help teach the course of laser principle. The complicated transverse modes of the symmetric confocal resonator can be visualized in personal computers, which is significant to help the students understand the pattern distribution of laser resonator.

Atmospheric propagation and combining of high-power lasers.

PubMed

Nelson, W; Sprangle, P; Davis, C C

2016-03-01

In this paper, we analyze beam combining and atmospheric propagation of high-power lasers for directed-energy (DE) applications. The large linewidths inherent in high-power fiber and slab lasers cause random phase and intensity fluctuations that occur on subnanosecond time scales. Coherently combining these high-power lasers would involve instruments capable of precise phase control and operation at rates greater than ∼10  GHz. To the best of our knowledge, this technology does not currently exist. This presents a challenging problem when attempting to phase lock high-power lasers that is not encountered when phase locking low-power lasers, for example, at milliwatt power levels. Regardless, we demonstrate that even if instruments are developed that can precisely control the phase of high-power lasers, coherent combining is problematic for DE applications. The dephasing effects of atmospheric turbulence typically encountered in DE applications will degrade the coherent properties of the beam before it reaches the target. Through simulations, we find that coherent beam combining in moderate turbulence and over multikilometer propagation distances has little advantage over incoherent combining. Additionally, in cases of strong turbulence and multikilometer propagation ranges, we find nearly indistinguishable intensity profiles and virtually no difference in the energy on the target between coherently and incoherently combined laser beams. Consequently, we find that coherent beam combining at the transmitter plane is ineffective under typical atmospheric conditions.

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...

Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model.

PubMed

Klemm, Matthias; Blum, Johannes; Link, Dietmar; Hammer, Martin; Haueisen, Jens; Schweitzer, Dietrich

2016-09-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes determined from the intensity decays. Here, we present a new technique to suppress the autofluorescence of the crystalline lens by introducing an annular stop into the detection light path, which we call Schweitzer's principle. The efficacy of annular stops with an outer diameter of 7 mm and inner diameters of 1 to 5 mm are analyzed in an experimental setup using a model eye based on fluorescent dyes. Compared to the confocal principle, Schweitzer's principle with an inner diameter of 3 mm is able to reduce the simulated crystalline lens fluorescence to 4%, while 42% of the simulated retina fluorescence is preserved. Thus, we recommend the implementation of Schweitzer's principle in scanning laser ophthalmoscopes used for fundus autofluorescence measurements, especially the FLIO device, for improved image quality.

Re-scan confocal microscopy: scanning twice for better resolution

PubMed Central

De Luca, Giulia M.R.; Breedijk, Ronald M.P.; Brandt, Rick A.J.; Zeelenberg, Christiaan H.C.; de Jong, Babette E.; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A.; Stallinga, Sjoerd; Manders, Erik M.M.

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required. PMID:24298422

Re-scan confocal microscopy: scanning twice for better resolution.

PubMed

De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver.

PubMed

Nakazawa, Yoshihisa; Takeda, Tsuyoshi; Suzuki, Nobuaki; Hayashi, Tatsushi; Harada, Yoko; Bamba, Takeshi; Kobayashi, Akio

2013-09-01

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.

Three-dimensional scanning confocal laser microscope

DOEpatents

Anderson, R. Rox; Webb, Robert H.; Rajadhyaksha, Milind

1999-01-01

A confocal microscope for generating an image of a sample includes a first scanning element for scanning a light beam along a first axis, and a second scanning element for scanning the light beam at a predetermined amplitude along a second axis perpendicular to the first axis. A third scanning element scans the light beam at a predetermined amplitude along a third axis perpendicular to an imaging plane defined by the first and second axes. The second and third scanning element are synchronized to scan at the same frequency. The second and third predetermined amplitudes are percentages of their maximum amplitudes. A selector determines the second and third predetermined amplitudes such that the sum of the percentages is equal to one-hundred percent.

A meta-analysis of confocal laser endomicroscopy for the detection of neoplasia in patients with Barrett's esophagus.

PubMed

Xiong, Yi-Quan; Ma, Shu-Juan; Zhou, Jun-Hua; Zhong, Xue-Shan; Chen, Qing

2016-06-01

Barrett's esophagus (BE) is considered the most important risk factor for development of esophageal adenocarcinoma. Confocal laser endomicroscopy (CLE) is a recently developed technique used to diagnose neoplasia in BE. This meta-analysis was performed to assess the accuracy of CLE for diagnosis of neoplasia in BE. We searched EMBASE, PubMed, Cochrane Library, and Web of Science to identify relevant studies for all articles published up to June 27, 2015 in English. The quality of included studies was assessed using QUADAS-2. Per-patient and per-lesion pooled sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio with 95% confidence intervals (CIs) were calculated. In total, 14 studies were included in the final analysis, covering 789 patients with 4047 lesions. Seven studies were included in the per-patient analysis. Pooled sensitivity and specificity were 89% (95% CI: 0.82-0.94) and 83% (95% CI: 0.78-0.86), respectively. Ten studies were included in the per-lesion analysis. Compared with the PP analysis, the corresponding pooled sensitivity declined to 77% (95% CI: 0.73-0.81) and specificity increased to 89% (95% CI: 0.87-0.90). Subgroup analysis showed that probe-based CLE (pCLE) was superior to endoscope-based CLE (eCLE) in pooled specificity [91.4% (95% CI: 89.7-92.9) vs 86.1% (95% CI: 84.3-87.8)] and AUC for the sROC (0.885 vs 0.762). Confocal laser endomicroscopy is a valid method to accurately differentiate neoplasms from non-neoplasms in BE. It can be applied to BE surveillance and early diagnosis of esophageal adenocarcinoma. © 2015 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Relationship between gustatory function and average number of taste buds per fungiform papilla measured by confocal laser scanning microscopy in humans.

PubMed

Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Manabe, Yasuhiro; Sano, Kazuo

2017-02-01

The aim of this study was to elucidate the relationship between the gustatory function and average number of taste buds per fungiform papilla (FP) in humans. Systemically healthy volunteers (n = 211), pre-operative patients with chronic otitis media (n = 79), and postoperative patients, with or without a chorda tympani nerve (CTN) severed during middle ear surgery (n = 63), were included. Confocal laser scanning microscopy was employed to observe fungiform taste buds because it allows many FP to be observed non-invasively in a short period of time. Taste buds in an average of 10 FP in the midlateral region of the tongue were counted. In total, 3,849 FP were observed in 353 subjects. The gustatory function was measured by electrogustometry (EGM). An inverse relationship was found between the gustatory function and average number of fungiform taste buds per papilla. The healthy volunteers showed a lower EGM threshold (better gustatory function) and had more taste buds than did the patients with otitis media, and the patients with otitis media showed a lower EGM threshold and had more taste buds than did postoperative patients, reflecting the severity of damage to the CTN. It was concluded that the confocal laser scanning microscope is a very useful tool for using to observe a large number of taste buds non-invasively. © 2017 Eur J Oral Sci.

Molecular confocal laser endomicroscopy: a novel technique for in vivo cellular characterization of gastrointestinal lesions.

PubMed

Karstensen, John Gásdal; Klausen, Pia Helene; Saftoiu, Adrian; Vilmann, Peter

2014-06-28

While flexible endoscopy is essential for macroscopic evaluation, confocal laser endomicroscopy (CLE) has recently emerged as an endoscopic method enabling visualization at a cellular level. Two systems are currently available, one based on miniprobes that can be inserted via a conventional endoscope or via a needle guided by endoscopic ultrasound. The second system has a confocal microscope integrated into the distal part of an endoscope. By adding molecular probes like fluorescein conjugated antibodies or fluorescent peptides to this procedure (either topically or systemically administered during on-going endoscopy), a novel world of molecular evaluation opens up. The method of molecular CLE could potentially be used for estimating the expression of important receptors in carcinomas, subsequently resulting in immediate individualization of treatment regimens, but also for improving the diagnostic accuracy of endoscopic procedures by identifying otherwise invisible mucosal lesions. Furthermore, studies have shown that fluorescein labelled drugs can be used to estimate the affinity of the drug to a target organ, which probably can be correlated to the efficacy of the drug. However, several of the studies in this research field have been conducted in animal facilities or in vitro, while only a limited number of trials have actually been carried out in vivo. Therefore, safety issues still needs further evaluations. This review will present an overview of the implications and pitfalls, as well as future challenges of molecular CLE in gastrointestinal diseases.

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...

Confocal laser-scanning microscopy of capillaries in normal and psoriatic skin

NASA Astrophysics Data System (ADS)

Archid, Rami; Patzelt, Alexa; Lange-Asschenfeldt, Bernhard; Ahmad, Sufian S.; Ulrich, Martina; Stockfleth, Eggert; Philipp, Sandra; Sterry, Wolfram; Lademann, Juergen

2012-10-01

An important and most likely active role in the pathogenesis of psoriasis has been attributed to changes in cutaneous blood vessels. The purpose of this study was to use confocal laser-scanning microscopy (CLSM) to investigate dermal capillaries in psoriatic and normal skin. The structures of the capillary loops in 5 healthy participants were compared with those in affected skin of 13 psoriasis patients. The diameters of the capillaries and papillae were measured for each group with CLSM. All investigated psoriasis patients showed elongated, widened, and tortuous microvessels in the papillary dermis, whereas all healthy controls showed a single capillary loop in each dermal papilla. The capillaries of the papillary loop and the dermal papilla were significantly enlarged in the psoriatic skin lesions (diameters 24.39±2.34 and 146.46±28.52 μm, respectively) in comparison to healthy skin (diameters 9.53±1.8 and 69.48±17.16 μm, respectively) (P<0.001). CLSM appears to represent a promising noninvasive technique for evaluating dermal capillaries in patients with psoriasis. The diameter of the vessels could be seen as a well-quantifiable indicator for the state of psoriatic skin. CLSM could be useful for therapeutic monitoring to delay possible recurrences.

A mercury arc lamp-based multi-color confocal real time imaging system for cellular structure and function.

PubMed

Saito, Kenta; Kobayashi, Kentaro; Tani, Tomomi; Nagai, Takeharu

2008-01-01

Multi-point scanning confocal microscopy using a Nipkow disk enables the acquisition of fluorescent images with high spatial and temporal resolutions. Like other single-point scanning confocal systems that use Galvano meter mirrors, a commercially available Nipkow spinning disk confocal unit, Yokogawa CSU10, requires lasers as the excitation light source. The choice of fluorescent dyes is strongly restricted, however, because only a limited number of laser lines can be introduced into a single confocal system. To overcome this problem, we developed an illumination system in which light from a mercury arc lamp is scrambled to make homogeneous light by passing it through a multi-mode optical fiber. This illumination system provides incoherent light with continuous wavelengths, enabling the observation of a wide range of fluorophores. Using this optical system, we demonstrate both the high-speed imaging (up to 100 Hz) of intracellular Ca(2+) propagation, and the multi-color imaging of Ca(2+) and PKC-gamma dynamics in living cells.

In Situ Observation of Kinetic Processes of Lath Bainite Nucleation and Growth by Laser Scanning Confocal Microscope in Reheated Weld Metals

NASA Astrophysics Data System (ADS)

Mao, Gaojun; Cao, Rui; Guo, Xili; Jiang, Yong; Chen, Jianhong

2017-12-01

The kinetic processes of nucleation and growth of bainite laths in reheated weld metals are observed and analyzed by a combination of a laser confocal scanning microscope and an electron backscattering diffraction with a field emission scanning electron microscope. The results indicate that the surface relief induced by phase transformation is able to reveal the real microstructural morphologies of bainite laths when viewed from various angles. Five nucleation modes and six types of growth behaviors of bainite laths are revealed. The bainite lath growth rates are measured to vary over a wide range, from 2 μm/s to higher than 2000 μm/s. The orientations of the bainite laths within a prior austenite grain are examined and denoted as different variants. On the basis of variant identification, the reason is analyzed for various growth rates which are demonstrated to be affected by (1) the density of the high-angle misorientation in it, (2) the included angle between habit planes of different variants, and (3) the direction of lath growth with respect to the free (polished) surface.

Use of portable devices and confocal Raman spectrometers at different wavelength to obtain the spectral information of the main organic components in tomato (Solanum lycopersicum) fruits.

PubMed

Trebolazabala, Josu; Maguregui, Maite; Morillas, Héctor; de Diego, Alberto; Madariaga, Juan Manuel

2013-03-15

Tomato (Solanum lycopersicum) fruit samples, in two ripening stages, ripe (red) and unripe (green), collected from a cultivar in the North of Spain (Barrika, Basque Country), were analyzed directly, without any sample pretreatment, with two different Raman instruments (portable spectrometer coupled to a micro-videocamera and a confocal Raman microscope), using two different laser excitation wavelengths (514 and 785 nm, only for the confocal microscope). The combined use of these laser excitation wavelengths allows obtaining, in a short period of time, the maximum spectral information about the main organic compounds present in this fruit. The major identified components of unripe tomatoes were cutin and cuticular waxes. On the other hand, the main components on ripe tomatoes were carotenes, polyphenoles and polysaccharides. Among the carotenes, it was possible to distinguish the presence of lycopene from β-carotene with the help of both excitation wavelengths, but specially using the 514 nm one, which revealed specific overtones and combination tones of this type of carotene. Copyright © 2012 Elsevier B.V. All rights reserved.

Aerogel Track Morphology: Measurement, Three Dimensional Reconstruction and Particle Location using Confocal Laser Scanning Microscopy

NASA Technical Reports Server (NTRS)

Kearsley, A. T.; Ball, A. D.; Wozniakiewicz, P. A.; Graham, G. A.; Burchell, M. J.; Cole, M. J.; Horz, F.; See, T. H.

2007-01-01

The Stardust spacecraft returned the first undoubted samples of cometary dust, with many grains embedded in the silica aerogel collector . Although many tracks contain one or more large terminal particles of a wide range of mineral compositions , there is also abundant material along the track walls. To help interpret the full particle size, structure and mass, both experimental simulation of impact by shots and numerical modeling of the impact process have been attempted. However, all approaches require accurate and precise measurement of impact track size parameters such as length, width and volume of specific portions. To make such measurements is not easy, especially if extensive aerogel fracturing and discoloration has occurred. In this paper we describe the application and limitations of laser confocal imagery for determination of aerogel track parameters, and for the location of particle remains.

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..

Two-dimensional confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

NASA Astrophysics Data System (ADS)

Jun, Brian; Giarra, Matthew; Golz, Brian; Main, Russell; Vlachos, Pavlos

2016-11-01

We present a methodology to mitigate the major sources of error associated with two-dimensional confocal laser scanning microscopy (CLSM) images of nanoparticles flowing through a microfluidic channel. The correlation-based velocity measurements from CLSM images are subject to random error due to the Brownian motion of nanometer-sized tracer particles, and a bias error due to the formation of images by raster scanning. Here, we develop a novel ensemble phase correlation with dynamic optimal filter that maximizes the correlation strength, which diminishes the random error. In addition, we introduce an analytical model of CLSM measurement bias error correction due to two-dimensional image scanning of tracer particles. We tested our technique using both synthetic and experimental images of nanoparticles flowing through a microfluidic channel. We observed that our technique reduced the error by up to a factor of ten compared to ensemble standard cross correlation (SCC) for the images tested in the present work. Subsequently, we will assess our framework further, by interrogating nanoscale flow in the cell culture environment (transport within the lacunar-canalicular system) to demonstrate our ability to accurately resolve flow measurements in a biological system.

Visualization and quantitation of abundant macroautophagy in virus-infected cells by confocal three-dimensional fluorescence imaging.

PubMed

Jackson, Wallen; Yamada, Masaki; Moninger, Thomas; Grose, Charles

2013-10-01

Varicella-zoster virus (VZV) is a human herpesvirus. Primary infection causes varicella (chickenpox), a viremic illness typified by an exanthem consisting of several hundred vesicles. When VZV reactivates from latency in the spinal ganglia during late adulthood, the emerging virus causes a vesicular dermatomal rash (herpes zoster or shingles). To expand investigations of autophagy during varicella and zoster, newer 3D imaging technology was combined with laser scanning confocal microscopy to provide animations of autophagosomes in the vesicular rash. First, the cells were immunolabeled with antibodies against VZV proteins and the LC3 protein, an integral autophagosomal protein. Antibody reagents lacking activity against the human blood group A1 antigen were selected. After laser excitation of the samples, optimized emission detection bandwidths were configured by Zeiss Zen control software. Confocal Z-stacks comprising up to 40 optical slices were reconstructed into 3D animations with the aid of Imaris software. With this imaging technology, individual autophagosomes were clearly detectable as spheres within each vesicular cell. To enumerate the number of autophagosomes, data sets from 50 cells were reconstructed as 3D fluorescence images and analyzed with MeasurementPro software. The mean number of autophagosomes per infected vesicular cell was >100, although over 200 autophagosomes were seen in a few cells. In summary, macroautophagy was easily quantitated within VZV-infected cells after immunolabeling and imaging by 3D confocal animation technology. These same 3D imaging techniques will be applicable for investigations of autophagy in other virus-infected cells. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Localization of puroindoline-a and lipids in bread dough using confocal scanning laser microscopy.

PubMed

Dubreil, Laurence; Biswas, Samares C; Marion, Didier

2002-10-09

Puroindolines are lipid-binding proteins from wheat flour that play a significant role in bread crumb texture. The localization of wheat flour lipids and puroindoline-a (PIN-a) in bread dough was studied by confocal scanning laser microscopy (CSLM). Wheat lipids were located around gas cells (GC) and embedded within the protein-starch matrix (SPM) of the dough. PIN-a was mainly located in the matrix of dough, where it was associated with lipids. In contrast, in defatted dough, PIN-a was found around GC. Addition of puroindolines in bread dough induced a defatting of the gas bubble surface and a decrease of the lipid vesicles and/or droplet size embedded within the SPM. Therefore, puroindolines control the lipid partitioning within the different phases of dough, a phenomenon that should have important consequence on the gas bubble expansion and GC formation in the further stages (fermentation, baking) of the bread-making process.

Modular Scanning Confocal Microscope with Digital Image Processing.

PubMed

Ye, Xianjun; McCluskey, Matthew D

2016-01-01

In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength.

Combination free electron and gaseous laser

DOEpatents

Brau, Charles A.; Rockwood, Stephen D.; Stein, William E.

1980-01-01

A multiple laser having one or more gaseous laser stages and one or more free electron stages. Each of the free electron laser stages is sequentially pumped by a microwave linear accelerator. Subsequently, the electron beam is directed through a gaseous laser, in the preferred embodiment, and in an alternative embodiment, through a microwave accelerator to lower the energy level of the electron beam to pump one or more gaseous lasers. The combination laser provides high pulse repetition frequencies, on the order of 1 kHz or greater, high power capability, high efficiency, and tunability in the synchronous production of multiple beams of coherent optical radiation.

Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

PubMed Central

Leigh, Steven Y.; Chen, Ye; Liu, Jonathan T.C.

2014-01-01

A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively ‘labels’ in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues. PMID:24940534

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. © Wiley Periodicals, Inc.

In situ detection of the Zn(2+) release process of ZnO NPs in tumour cells by confocal laser scanning fluorescence microscopy.

PubMed

Song, Wenshuang; Tang, Xiaoling; Li, Yong; Sun, Yang; Kong, Jilie; Qingguang, Ren

2016-08-01

The use of zinc oxide (ZnO) nanoparticles (NPs) for cancer is not yet clear for human clinical applications, which is primarily due to the lack of a better understanding of the action mechanisms and cellular consequences of the direct exposure of cells to these NPs. In this work, the authors have selected zinquin ethyl ester, a Zn(2+)-specific fluorescent molecular probe, to efficiently differentiate ZnO NPs and Zn(2+), and combined with confocal laser scanning microscopy (CLSM) to in situ study the Zn(2+) release process of ZnO NPs in cancer cell system through detecting the change of Zn(2+) level over time. During the experiments, the authors have designed the test group ZnO-2 in addition to assess the influence of a long-term storage on the characteristics of ZnO NPs in aqueous solution, and the Zn(2+) release process of ZnO NPs in cancer cell system. After three-month storage at room temperature, the release process became earlier and faster, which was consistent with previous results of transmission electron microscope, UV-Vis and PL spectra. It is a good detection method that combination of Zn(2+)-specific fluorescent molecular probe and CLSM, which will be helpful for ZnO NPs using in clinical research.

Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model

PubMed Central

Klemm, Matthias; Blum, Johannes; Link, Dietmar; Hammer, Martin; Haueisen, Jens; Schweitzer, Dietrich

2016-01-01

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes determined from the intensity decays. Here, we present a new technique to suppress the autofluorescence of the crystalline lens by introducing an annular stop into the detection light path, which we call Schweitzer’s principle. The efficacy of annular stops with an outer diameter of 7 mm and inner diameters of 1 to 5 mm are analyzed in an experimental setup using a model eye based on fluorescent dyes. Compared to the confocal principle, Schweitzer’s principle with an inner diameter of 3 mm is able to reduce the simulated crystalline lens fluorescence to 4%, while 42% of the simulated retina fluorescence is preserved. Thus, we recommend the implementation of Schweitzer’s principle in scanning laser ophthalmoscopes used for fundus autofluorescence measurements, especially the FLIO device, for improved image quality. PMID:27699092

Testing the Crystalline Integrity of Baddeleyite: A Systematic EBSD and Confocal Laser-Raman Spectroscopy Study

NASA Astrophysics Data System (ADS)

DesOrmeau, J. W.; Ibanez-Mejia, M.; Lafuente Valverde, B.; Eddy, M. P.; Trail, D.; Wang, Y.

2017-12-01

The accessory mineral baddeleyite (monoclinic ZrO2) has great potential as a high-precision U-Pb geochronometer in silica-undersaturated rocks. However, due to a lack of understanding of alpha-recoil damage to the crystal structure and the influences on chemical diffusion properties and closed-system behavior, limitations still exist. Studies have shown phase transformations in baddeleyite (monoclinic to tetragonal) resulting from radiation damage due to ion bombardment [e.g., 1], but the effects of self-irradiation on the baddeleyite crystal structure over geologic timescales remain poorly understood. A recent study reported confocal laser-Raman spectra suggesting the presence of a tetragonal phase in a Phalaborwa baddeleyite [2], which has crucial implications for Pb mobility and high-precision U-Pb results. To better understand the physical effects of self-irradiation in baddeleyite over geologic timescales, samples of various ages (ca. 2060-32 Ma) and calculated alpha-doses (0.001-0.901 x 1016 α/mg) were imaged by cathodoluminescence (CL) and subsequently analyzed via electron backscatter diffraction (EBSD) and confocal laser-Raman spectroscopy. Synthetic baddeleyite grown in a one-atmosphere furnace was also analyzed to allow a comparison of EBSD and Raman results of the pure monoclinic phase and the natural unknown samples. Whole grain EBSD maps (n=75) of baddeleyite from the Phalabowra and Kovdor carbonatites, the Ammänpelto sill, and the Duluth, Ogden and Yinmawanshan gabbros show complex twinning that is loosely correlated to CL zoning and identification of the monoclinic phase only, suggesting no evidence for phase transformations within grains of contrasting age and amounts of alpha-doses. Preliminary Raman results show evidence of systematic shifts in the vibrational modes of Phalabowra baddeleyite with respect to the younger crystals, which may be the result of radiation-induced damage accumulation rather than phase transformation. These results aim

Three-photon fluorescence imaging of melanin with a dual-wedge confocal scanning system

NASA Astrophysics Data System (ADS)

Mega, Yair; Kerimo, Joseph; Robinson, Joseph; Vakili, Ali; Johnson, Nicolette; DiMarzio, Charles

2012-03-01

Confocal microscopy can be used as a practical tool in non-invasive applications in medical diagnostics and evaluation. In particular, it is being used for the early detection of skin cancer to identify pathological cellular components and, potentially, replace conventional biopsies. The detection of melanin and its spatial location and distribution plays a crucial role in the detection and evaluation of skin cancer. Our previous work has shown that the visible emission from melanin is strong and can be easily observed with a near-infrared CW laser using low power. This is due to a unique step-wise, (SW) three-photon excitation of melanin. This paper shows that the same SW, 3-photon fluorescence can also be achieved with an inexpensive, continuous-wave laser using a dual-prism scanning system. This demonstrates that the technology could be integrated into a portable confocal microscope for clinical applications. The results presented here are in agreement with images obtained with the larger and more expensive femtosecond laser system used earlier.

Quantitative and Qualitative Aspects of Gas-Metal-Oxide Mass Transfer in High-Temperature Confocal Scanning Laser Microscopy

NASA Astrophysics Data System (ADS)

Piva, Stephano P. T.; Pistorius, P. Chris; Webler, Bryan A.

2018-05-01

During high-temperature confocal scanning laser microscopy (HT-CSLM) of liquid steel samples, thermal Marangoni flow and rapid mass transfer between the sample and its surroundings occur due to the relatively small sample size (diameter around 5 mm) and large temperature gradients. The resulting evaporation and steel-slag reactions tend to change the chemical composition in the metal. Such mass transfer effects can change observed nonmetallic inclusions. This work quantifies oxide-metal-gas mass transfer of solutes during HT-CSLM experiments using computational simulations and experimental data for (1) dissolution of MgO inclusions in the presence and absence of slag and (2) Ca, Mg-silicate inclusion changes upon exposure of a Si-Mn-killed steel to an oxidizing gas atmosphere.

In vivo analysis of THz wave irradiation induced acute inflammatory response in skin by laser-scanning confocal microscopy.

PubMed

Hwang, Yoonha; Ahn, Jinhyo; Mun, Jungho; Bae, Sangyoon; Jeong, Young Uk; Vinokurov, Nikolay A; Kim, Pilhan

2014-05-19

The recent development of THz sources in a wide range of THz frequencies and power levels has led to greatly increased interest in potential biomedical applications such as cancer and burn wound diagnosis. However, despite its importance in realizing THz wave based applications, our knowledge of how THz wave irradiation can affect a live tissue at the cellular level is very limited. In this study, an acute inflammatory response caused by pulsed THz wave irradiation on the skin of a live mouse was analyzed at the cellular level using intravital laser-scanning confocal microscopy. Pulsed THz wave (2.7 THz, 4 μs pulsewidth, 61.4 μJ per pulse, 3Hz repetition), generated using compact FEL, was used to irradiate an anesthetized mouse's ear skin with an average power of 260 mW/cm(2) for 30 minutes using a high-precision focused THz wave irradiation setup. In contrast to in vitro analysis using cultured cells at similar power levels of CW THz wave irradiation, no temperature change at the surface of the ear skin was observed when skin was examined with an IR camera. To monitor any potential inflammatory response, resident neutrophils in the same area of ear skin were repeatedly visualized before and after THz wave irradiation using a custom-built laser-scanning confocal microscopy system optimized for in vivo visualization. While non-irradiated control skin area showed no changes in the number of resident neutrophils, a massive recruitment of newly infiltrated neutrophils was observed in the THz wave irradiated skin area after 6 hours, which suggests an induction of acute inflammatory response by the pulsed THz wave irradiation on the skin via a non-thermal process.

[The combined laser therapy of rheumatoid arthritis].

PubMed

Sidorov, V D; Mamiliaeva, D R; Derevnina, N A; Reformatskaia, S Iu

2000-01-01

Low-intensity infrared laser radiation to the tympanic vessels was studied as one of the hemophysiotherapeutic methods and as a component of combined treatment in which it accompanies local transcutaneous laser radiation of the affected joints. It is shown that immunomodulation is feasible under noninvasive interauricular laser effect on hemostasis. Indications for both laser regimens are formulated. Joint exposure to transcutaneous laser radiation is contraindicated if the affected joints have an exudative component of inflammation.

Efficiency of the confocal method of laser endomicroscopy in complex diagnoses of diseases of common bile duct

NASA Astrophysics Data System (ADS)

Anaskin, S. G.; Panchenkov, D. N.; Chertyuk, V. B.; Sazonov, D. V.; Zabozlayev, F. G.; Danilevskaya, O. V.; Mokshina, N. V.; Korniletsky, I. D.

2017-01-01

One of the more frequent manifestations of diseases of the bile ducts are its’ strictures or stenoses that could be of either malignant or benign nature. Current methods of diagnosing this pathology include computer tomography (CT) scan, magnetic resonance cholangiopancreatography (MRCP), endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP). However, these methods are not always informative, which makes this a current and topical problem. A fundamentally new method that broadens the capabilities of ERCP when diagnosing diseases of the bile duct accompanied by the development of strictures or stenoses is probe-based confocal laser endomicroscopy (pCLE). The method is based on the principle of confocal fluorescence microscopy. The most elaborate complications arise with the presence of the pre-existing pancreatobiliary pathology: pseudotumoral chronic pancreatitis, acute cholangitis, etc. Early stage cholangiocarcinoma diagnosis can be difficult (and not always possible) even with the help of modern research methods. For the timely diagnostic it is advantageous to conduct pCLE and targeted biopsy of the zone with most manifested changes. In all instances, the first use of the pCLE method for diagnostic purposes allowed us to clarify and correctly verify the diagnosis. When concerning the diseases of the bile duct, the modern stage of pCLE development can be of critical importance when other methods are not effective.

Elastomeric photo-actuators and their investigation by confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Czaniková, Klaudia; Ilčíková, Markéta; Krupa, Igor; Mičušík, Matej; Kasák, Peter; Pavlova, Ewa; Mosnáček, Jaroslav; Chorvát, Dušan, Jr.; Omastová, Mária

2013-10-01

The photo-actuation behavior of nanocomposites based on ethylene-vinylacetate copolymer (EVA) and styrene-isoprene-styrene (SIS) block copolymer filled with well-dispersed and modified multiwalled carbon nanotubes (MWCNTs) is discussed in this paper. The nanocomposites were prepared by casting from solution. To improve the dispersion of the MWCNTs in EVA, the MWCNT surface was modified with a non-covalent surfactant, cholesteryl 1-pyrenecarboxylate (PyChol). To prepare SIS nanocomposites, the MWCNT surface was covalently modified with polystyrene chains. The good dispersion of the filler was confirmed by transmission electron microscopy (TEM). Special, custom-made punch/die molds were used to create a Braille element (BE)-like shape, which under shear forces induces a uniaxial orientation of the MWCNTs within the matrix. The uniaxial orientation of MWCNTs is an essential precondition to ensure the photo-actuating behavior of MWCNTs in polymeric matrices. The orientation of the MWCNTs within the matrices was examined by scanning electron microscopy (SEM). Nanocomposite BEs were illuminated from the bottom by a red light-emitting diode (LED), and the photo-actuation was investigated by confocal laser scanning microscopy (CLSM). When the BEs were exposed to light, a temporary increase in the height of the element was detected. This process was observed to be reversible: after switching off the light, the BEs returned to their original shape and height.

Characterization of a subwavelength-scale 3D void structure using the FDTD-based confocal laser scanning microscopic image mapping technique.

PubMed

Choi, Kyongsik; Chon, James W; Gu, Min; Lee, Byoungho

2007-08-20

In this paper, a simple confocal laser scanning microscopic (CLSM) image mapping technique based on the finite-difference time domain (FDTD) calculation has been proposed and evaluated for characterization of a subwavelength-scale three-dimensional (3D) void structure fabricated inside polymer matrix. The FDTD simulation method adopts a focused Gaussian beam incident wave, Berenger's perfectly matched layer absorbing boundary condition, and the angular spectrum analysis method. Through the well matched simulation and experimental results of the xz-scanned 3D void structure, we first characterize the exact position and the topological shape factor of the subwavelength-scale void structure, which was fabricated by a tightly focused ultrashort pulse laser. The proposed CLSM image mapping technique based on the FDTD can be widely applied from the 3D near-field microscopic imaging, optical trapping, and evanescent wave phenomenon to the state-of-the-art bio- and nanophotonics.

High-power direct diode laser output by spectral beam combining

NASA Astrophysics Data System (ADS)

Tan, Hao; Meng, Huicheng; Ruan, Xu; Du, Weichuan; Wang, Zhao

2018-03-01

We demonstrate a spectral beam combining scheme based on multiple mini-bar stacks, which have more diode laser combining elements, to increase the combined diode laser power and realize equal beam quality in both the fast and slow axes. A spectral beam combining diode laser output of 1130 W is achieved with an operating current of 75 A. When a 9.6 X de-magnifying telescope is introduced between the output mirror and the diffraction grating, to restrain cross-talk among diode laser emitters, a 710 W spectral beam combining diode laser output is achieved at the operating current of 70 A, and the beam quality on the fast and slow axes of the combined beam is about 7.5 mm mrad and 7.3 mm mrad respectively. The power reduction is caused by the existence of a couple resonator between the rear facet of the diode laser and the fast axis collimation lens, and it should be eliminated by using diode laser chips with higher front facet transmission efficiency and a fast axis collimation lens with lower residual reflectivity.

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy.

PubMed

Boyde, A; Vesely, P; Gray, C; Jones, S J

1994-01-01

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37 degrees C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.

Confocal Imaging of porous media

NASA Astrophysics Data System (ADS)

Shah, S.; Crawshaw, D.; Boek, D.

2012-12-01

Carbonate rocks, which hold approximately 50% of the world's oil and gas reserves, have a very complicated and heterogeneous structure in comparison with sandstone reservoir rock. We present advances with different techniques to image, reconstruct, and characterize statistically the micro-geometry of carbonate pores. The main goal here is to develop a technique to obtain two dimensional and three dimensional images using Confocal Laser Scanning Microscopy. CLSM is used in epi-fluorescent imaging mode, allowing for the very high optical resolution of features well below 1μm size. Images of pore structures were captured using CLSM imaging where spaces in the carbonate samples were impregnated with a fluorescent, dyed epoxy-resin, and scanned in the x-y plane by a laser probe. We discuss the sample preparation in detail for Confocal Imaging to obtain sub-micron resolution images of heterogeneous carbonate rocks. We also discuss the technical and practical aspects of this imaging technique, including its advantages and limitation. We present several examples of this application, including studying pore geometry in carbonates, characterizing sub-resolution porosity in two dimensional images. We then describe approaches to extract statistical information about porosity using image processing and spatial correlation function. We have managed to obtain very low depth information in z -axis (~ 50μm) to develop three dimensional images of carbonate rocks with the current capabilities and limitation of CLSM technique. Hence, we have planned a novel technique to obtain higher depth information to obtain high three dimensional images with sub-micron resolution possible in the lateral and axial planes.

All-fiber 7x1 signal combiner for incoherent laser beam combining

NASA Astrophysics Data System (ADS)

Noordegraaf, D.; Maack, M. D.; Skovgaard, P. M. W.; Johansen, J.; Becker, F.; Belke, S.; Blomqvist, M.; Laegsgaard, J.

2011-02-01

We demonstrate an all-fiber 7x1 signal combiner for incoherent laser beam combining. This is a potential key component for reaching several kW of stabile laser output power. The combiner couples the output from 7 single-mode (SM) fiber lasers into a single multi-mode (MM) fiber. The input signal fibers have a core diameter of 17 μm and the output MM fiber has a core diameter of 100 μm. In a tapered section light gradually leaks out of the SM fibers and is captured by a surrounding fluorine-doped cladding. The combiner is tested up to 2.5 kW of combined output power and only a minor increase in device temperature is observed. At an intermediate power level of 600 W a beam parameter product (BPP) of 2.22 mm x mrad is measured, corresponding to an M2 value of 6.5. These values are approaching the theoretical limit dictated by brightness conservation.

Modular Scanning Confocal Microscope with Digital Image Processing

PubMed Central

McCluskey, Matthew D.

2016-01-01

In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength. PMID:27829052

Handheld confocal Raman microspectrometer for in-vivo skin cancer measurement

NASA Astrophysics Data System (ADS)

Lieber, Chad A.; Ellis, Darrel L.; Billheimer, D. D.; Mahadevan-Jansen, Anita

2004-07-01

Several studies have demonstrated Raman spectroscopy to be capable of tissue diagnosis with accuracy rivaling that of histopathologic analysis. This technique obtains biochemical-specific information noninvasively, and can eliminate the pain, time, and cost associated with biopsy and pathological analysis. Furthermore, when used in a confocal arrangement, Raman spectra can be obtained from localized regions of the tissue. Skin cancers are an ideal candidate for this emerging technology, due to their obvious accessibility and presentation at specific depths. However, most commercially available confocal Raman microspectrometers are large, rigid systems ill-suited for clinical application. We developed a bench-top confocal Raman microspectrometer using a portable external-cavity diode laser excitation source. This system was used to study several skin lesions in vitro. Results show the depth-resolved Raman spectra can diagnose in vitro skin lesions with 96% sensitivity, 88% specificity, and 86% pathological classification accuracy. Based on the success of this study, a portable Raman system with a handheld confocal microscope was developed for clinical application. Preliminary in vivo data show several distinct spectral differences between skin pathologies. Diagnostic algorithms are planned for this continuing study to assess the capability of Raman spectroscopy for clinical skin cancer diagnosis.

Confocal laser endomicroscopy for in vivo diagnosis of Barrett's oesophagus and associated neoplasia: a pilot study conducted in a single Italian centre.

PubMed

Trovato, Cristina; Sonzogni, Angelica; Ravizza, Davide; Fiori, Giancarla; Tamayo, Darina; De Roberto, Giuseppe; de Leone, Annalisa; De Lisi, Stefania; Crosta, Cristiano

2013-05-01

Diagnosis and management of Barrett's oesophagus are controversial. Technical improvements in real-time recognition of intestinal metaplasia and neoplastic foci provide the chance for more effective target biopsies. Confocal laser endomicroscopy allows to analyze living cells during endoscopy. To assess the diagnostic accuracy, inter- and intra-observer variability of endomicroscopy for detecting in vivo neoplasia (dysplasia and/or early neoplasia) in Barrett's oesophagus. Prospective pilot study. Patients referred for known Barrett's oesophagus were screened. Endomicroscopy was carried out in a circular fashion, every 1-2 cm, on the whole columnar-lined distal oesophagus. Visible lesions, when present, were analyzed first. Targeted biopsies were taken. Confocal images were classified according to confocal Barrett classification. Endomicroscopic and histological findings were compared. Forty-eight out of 50 screened patients underwent endomicroscopy. Visible lesions were observed in 3 patients. In a per-biopsy analysis, Barrett's-oesophagus-associated neoplasia could be predicted with an accuracy of 98.1%. The agreement between endomicroscopic and histological results was substantial (κ=0.76). This study suggests that endomicroscopy can provide in vivo diagnosis of Barrett's oesophagus-associated neoplasia. Because it allows for the study of larger surface areas of the mucosa, endomicroscopy may lead to significant improvements in the in vivo screening and surveillance of Barrett's oesophagus. Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Atmospheric propagation and combining of high power lasers: comment.

PubMed

Goodno, Gregory D; Rothenberg, Joshua E

2016-10-10

Nelson et al. [Appl. Opt.55, 1757 (2016)APOPAI0003-693510.1364/AO.55.001757] recently concluded that coherent beam combining and remote phase locking of high-power lasers are fundamentally limited by the laser source linewidth. These conclusions are incorrect and not relevant to practical high-power coherently combined laser architectures.

Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system

PubMed Central

Kelner, Roy; Katz, Barak; Rosen, Joseph

2015-01-01

We propose a new type of confocal microscope using Fresnel incoherent correlation holography (FINCH). Presented here is a confocal configuration of FINCH using a phase pinhole and point illumination that is able to suppress out-of-focus information from the recorded hologram and hence combine the super-resolution capabilities of FINCH with the sectioning capabilities of confocal microscopy. PMID:26413560

Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging.

PubMed

Schneider, Falk; Waithe, Dominic; Galiani, Silvia; Bernardino de la Serna, Jorge; Sezgin, Erdinc; Eggeling, Christian

2018-06-19

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED-FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED-FCS measurement method, line interleaved excitation scanning STED-FCS (LIESS-FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS-FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS-FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Application of Laser Scanning Confocal Microscopy to Heat and Mass Transport Modeling in Porous Microstructures

NASA Technical Reports Server (NTRS)

Marshall, Jochen; Milos, Frank; Fredrich, Joanne; Rasky, Daniel J. (Technical Monitor)

1997-01-01

Laser Scanning Confocal Microscopy (LSCM) has been used to obtain digital images of the complicated 3-D (three-dimensional) microstructures of rigid, fibrous thermal protection system (TPS) materials. These orthotropic materials are comprised of refractory ceramic fibers with diameters in the range of 1 to 10 microns and have open porosities of 0.8 or more. Algorithms are being constructed to extract quantitative microstructural information from the digital data so that it may be applied to specific heat and mass transport modeling efforts; such information includes, for example, the solid and pore volume fractions, the internal surface area per volume, fiber diameter distributions, and fiber orientation distributions. This type of information is difficult to obtain in general, yet it is directly relevant to many computational efforts which seek to model macroscopic thermophysical phenomena in terms of microscopic mechanisms or interactions. Two such computational efforts for fibrous TPS materials are: i) the calculation of radiative transport properties; ii) the modeling of gas permeabilities.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

PubMed

Pucci, Angela; Mattioli, Claudia; Matteucci, Marco; Lorenzini, Daniele; Panvini, Francesca; Pacini, Simone; Ippolito, Chiara; Celiento, Michele; De Martino, Andrea; Dolfi, Amelio; Belgio, Beatrice; Bortolotti, Uberto; Basolo, Fulvio; Bartoloni, Giovanni

2018-05-22

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

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...

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...

Polarization/Spatial Combining of Laser-Diode Pump Beams

NASA Technical Reports Server (NTRS)

Gelsinger, Paul; Liu, Duncan

2008-01-01

A breadboard version of an optical beam combiner is depicted which make it possible to use the outputs of any or all of four multimode laser diodes to pump a non-planar ring oscillator (NPRO) laser. The output of each laser diode has a single-mode profile in the meridional plane containing an axis denoted the 'fast' axis and a narrower multimode profile in the orthogonal meridional plane, which contains an axis denoted the 'slow' axis and a narrower multimode profile in the orthogonal meridional plane, which contains an axis denoted the 'slow' axis. One of the purposes served by the beam-combining optics is to reduce the fast-axis numerical aperture (NA) of the laser-diode output to match the NA of the optical fiber. Along the slow axis, the unmodified laser-diode NA is already well matched to the fiber optic NA, so no further slow-axis beam shaping is needed. In this beam combiner, the laser-diode outputs are collimated by aspherical lenses, then half-wave plates and polarizing beam splitters are used to combine the four collimated beams into two beams. Spatial combination of the two beams and coupling into the optical fiber is effected by use of anamorphic prisms, mirrors, and a focusing lens. The anamorphic prisms are critical elements in the NA-matching scheme, in that they reduce the fast-axis beam width to 1/6 of its original values. Inasmuch as no slow-axis beam shaping is needed, the collimating and focusing lenses are matched for 1:1 iumaging. Because these lenses are well corrected for infinite conjugates the combiner offers diffraction-limited performance along both the fast and slow axes.

Confocal analysis of the exopolysaccharide matrix of Candida albicans biofilms.

PubMed

Gonçalves, Letícia M; Del Bel Cury, Altair A; de Vasconcellos, Andréa A; Cury, Jaime A; da Silva, Wander J

2015-08-01

Confocal laser-scanning microscopy (CLSM) was carried out to investigate the exopolysaccharide matrix of Candida albicans (C. albicans) biofilms developed on denture material under dietary carbohydrate exposure. Biofilms were developed on poly(methyl methacrylate) discs in culture media without (control) or with supplementation by glucose or sucrose for 72 h. For the CLSM analysis, biofilms were labeled with concanavalin A (ConA) during its development. Afterwards, biofilms were also labeled with SYTO-9. To confirm the results, the matrix was investigated by the phenol-sulfuric method. Data were analyzed by anova, followed by Tukey's test, with the level of significance set at 5%. The use of ConA during biofilm development provided effective labeling of the exopolysaccharide matrix. The exposure to sucrose resulted in biofilms with the highest exopolysaccharide matrix biovolume (P < 0.05). The characterization obtained by CLSM was confirmed by the phenol-sulfuric method. Confocal laser-scanning microscopy was found to be an effective tool for investigating the exopolysaccharide matrix of C. albicans biofilms, and exposure to sucrose resulted in increased matrix production. © 2014 Wiley Publishing Asia Pty Ltd.

Effect of chitosan nanoparticle, QMix, and EDTA on TotalFill BC sealers' dentinal tubule penetration: a confocal laser scanning microscopy study.

PubMed

Aydın, Zeliha Uğur; Özyürek, Taha; Keskin, Büşra; Baran, Talat

2018-04-12

The aim of the present study was to compare the effect of chitosan nanoparticle, QMix, and 17% EDTA on the penetrability of a calcium silicate-based sealer into dentinal tubules using a confocal laser scanning microscope (CLSM). Sixty mandibular premolar teeth were selected and randomly divided into three groups (n = 20) before root canal preparation according to the solution used in the final rinse protocol: chitosan, QMix, and EDTA groups. Twenty teeth of each group were filled with a TotalFill BC sealers' single gutta-percha cone and with 0.1% rhodamine B. The specimens were horizontally sectioned at 3 and 5 mm from the apex, and the slices were analyzed in CLSM (4×). Total percentage and maximum depth of sealer penetration were measured using confocal laser scanning microscopy with using Image J analysis software. Dentinal tubule's penetration depth, percentage, and area were measured using imaging software. Kruskal-Wallis test was used for statistical analysis. The level of significance was set at 5%. Results of Kruskal-Wallis analysis showed that there was a significant difference in the percentage and depth of sealer penetration among all groups at 3 and 5 mm level sections (P < 0.05). Within the groups, the minimum sealer penetration depth was recorded for chitosan nanoparticle group. Greater depth of sealer penetration was recorded at 5 mm as compared to 3 mm in all the groups. Within the limitation of the present study, it can be concluded that QMix and EDTA promoted sealer penetration superior to that achieved by chitosan nanoparticle.

The learning curve, interobserver, and intraobserver agreement of endoscopic confocal laser endomicroscopy in the assessment of mucosal barrier defects.

PubMed

Chang, Jeff; Ip, Matthew; Yang, Michael; Wong, Brendon; Power, Theresa; Lin, Lisa; Xuan, Wei; Phan, Tri Giang; Leong, Rupert W

2016-04-01

Confocal laser endomicroscopy can dynamically assess intestinal mucosal barrier defects and increased intestinal permeability (IP). These are functional features that do not have corresponding appearance on histopathology. As such, previous pathology training may not be beneficial in learning these dynamic features. This study aims to evaluate the diagnostic accuracy, learning curve, inter- and intraobserver agreement for identifying features of increased IP in experienced and inexperienced analysts and pathologists. A total of 180 endoscopic confocal laser endomicroscopy (Pentax EC-3870FK; Pentax, Tokyo, Japan) images of the terminal ileum, subdivided into 6 sets of 30 were evaluated by 6 experienced analysts, 13 inexperienced analysts, and 2 pathologists, after a 30-minute teaching session. Cell-junction enhancement, fluorescein leak, and cell dropout were used to represent increased IP and were either present or absent in each image. For each image, the diagnostic accuracy, confidence, and quality were assessed. Diagnostic accuracy was significantly higher for experienced analysts compared with inexperienced analysts from the first set (96.7% vs 83.1%, P < .001) to the third set (95% vs 89.7, P = .127). No differences in accuracy were noted between inexperienced analysts and pathologists. Confidence (odds ratio, 8.71; 95% confidence interval, 5.58-13.57) and good image quality (odds ratio, 1.58; 95% confidence interval, 1.22-2.03) were associated with improved interpretation. Interobserver agreement κ values were high and improved with experience (experienced analysts, 0.83; inexperienced analysts, 0.73; and pathologists, 0.62). Intraobserver agreement was >0.86 for experienced observers. Features representative of increased IP can be rapidly learned with high inter- and intraobserver agreement. Confidence and image quality were significant predictors of accurate interpretation. Previous pathology training did not have an effect on learning. Copyright © 2016

In vivo confocal Raman spectroscopy of the human cornea.

PubMed

Bauer, N J; Hendrikse, F; March, W F

1999-07-01

To investigate the feasibility of a confocal Raman spectroscopic technique for the noninvasive assessment of corneal hydration in vivo in two legally blind subjects. A laser beam (632.8 nm; 15 mJ) was maintained on the cornea by using a microscope objective lens (x25 magnification, NA = 0.5, f = 10 mm) both for focusing the incident light as well as collecting the Raman backscattered light, in a 180 degrees backscatter configuration. An optical fiber, acting as the confocal pinhole for elimination of light from out-of-focus places, was coupled to a spectrometer that dispersed the collected light onto a sensitive array detector for rapid spectral data acquisition over a range from 2,890 to 3,590/cm(-1). Raman spectra were recorded from the anterior 100-150 microm of the cornea over a period before and after topical application of a mild dehydrating solution. The ratio between the amplitudes of the signals at 3,400/cm(-1) (OH-vibrational mode of water) and 2,940/cm(-1) (CH-vibrational mode of proteins) was used as a measure for corneal hydration. High signal-to-noise ratio (SNR = 25) Raman spectra were obtained from the human corneas by using 15 mJ of laser light energy. Qualitative changes in the hydration of the anteriormost part of the corneas could be observed as a result of the dehydrating agent. With adequate improvements in system safety, confocal Raman spectroscopy could potentially be applied clinically as a noninvasive tool for the assessment of corneal hydration in vivo.

Analysis of protein and lipid dynamics using confocal fluorescence recovery after photobleaching (FRAP)

PubMed Central

Day, Charles A.; Kraft, Lewis J.; Kang, Minchul; Kenworthy, Anne K.

2012-01-01

Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection and analysis. In this review we discuss the theoretical basis for confocal FRAP, followed by step-by-step protocols for FRAP data acquisition using a laser scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented. PMID:23042527

Determination of the Subcellular Distribution of Liposomes Using Confocal Microscopy.

PubMed

Solomon, Melani A

2017-01-01

It is being increasingly recognized that therapeutics need to be delivered to specific organelle targets within cells. Liposomes are versatile lipid-based drug delivery vehicles that can be surface-modified to deliver the loaded cargo to specific subcellular locations within the cell. Hence, the development of such technology requires a means of measuring the subcellular distribution possibly by utilizing imaging techniques that can visualize and quantitate the extent of this subcellular localization. The apparent increase of resolution along the Z-axis offered by confocal microscopy makes this technique suitable for such studies. In this chapter, we describe the application of confocal laser scanning microscopy (CLSM) to determine the subcellular distribution of fluorescently labeled mitochondriotropic liposomes.

Iris ultrastructure in patients with synechiae as revealed by in vivo laser scanning confocal microscopy : In vivo iris ultrastructure in patients with Synechiae by Laser Scanning Confocal Microscopy.

PubMed

Li, Ming; Cheng, Hongbo; Guo, Ping; Zhang, Chun; Tang, Song; Wang, Shusheng

2016-04-26

Iris plays important roles in ocular physiology and disease pathogenesis. Currently it is technically challenging to noninvasively examine the human iris ultrastructure in vivo. The purpose of the current study is to reveal human iris ultrastructure in patients with synechiae by using noninvasive in vivo laser scanning confocal microscopy (LSCM). The ultrastructure of iris in thirty one patients, each with synechiae but transparent cornea, was examined by in vivo LSCM. Five characteristic iris ultrastructures was revealed in patients with synechiae by in vivo LSCM, which include: 1. tree trunk-like structure; 2. tree branch/bush-like structure; 3. Fruit-like structure; 4. Epithelioid-like structure; 5. deep structure. Pigment granules can be observed as a loose structure on the top of the arborization structure. In iris-associated diseases with Tyndall's Phenomenon and keratic precipitates, the pigment particles are more likely to fall off from the arborization structure. The ultrastructure of iris in patients with synechiae has been visualized using in vivo LSCM. Five iris ultrastructures can be clearly observed, with some of the structures maybe disease-associated. The fall-off of the pigment particles may cause the Tyndall's Phenomenon positive. In vivo LSCM provides a non-invasive approach to observe the human iris ultrastructure under certain eye disease conditions, which sets up a foundation to visualize certain iris-associated diseases in the future.

Confocal endomicroscopy of the larynx

NASA Astrophysics Data System (ADS)

Just, T.; Wiechmann, T.; Stachs, O.; Stave, J.; Guthoff, R.; Hüttmann, G.; Pau, H. W.

2012-02-01

Beside the good image quality with the confocal laser scanning microscope (HRTII) and the Rostock Cornea Module (RCM), this technology can not be used to investigate the human larynx in vivo. To accomplish this, a rigid custom-made endoscope (KARL STORZ GmbH & Co. KG; Tuttlingen Germany) was developed. A connector was developed to connect the scanner head of the HRTII to the rigid endoscope. With the connector, the starting plane can be set manually. To achieve optical sectioning of the laryngeal tissue (80 μm per volume scan), the scanning mechanism of the HRTII needs to be activated using a foot switch. The devices consisting of the endoscope, HRTII, and the connector supply images of 400 x 400 μm and reach average penetration depths of 100-300 μm (λ/4 plate of the scanner head of the HRTII was removed). The lateral and axial resolutions are about 1-2 μm and 2 μm, respectively. In vivo rigid confocal endoscopy is demonstrated with an acquisition time for a volume scan of 6 s. The aim of this study was to differentiate pre-malignant laryngeal lesions from micro-invasive carcinoma of the larynx. 22 patients with suspicious lesions of the true vocal cords were included. This pilot study clearly demonstrates the possibility to detect dysplastic cells close to the basal cell layer and within the subepithelial space in lesions with small leukoplakia (thin keratin layer). These findings may have an impact on microlaryngoscopy to improve the precision for biopsy and on microlaryngoscopic laser surgery of the larynx to identify the margins of the pre-malignant lesion.

Sealer penetration into dentinal tubules in the presence or absence of smear layer: a confocal laser scanning microscopic study.

PubMed

Kuçi, Astrit; Alaçam, Tayfun; Yavaş, Ozer; Ergul-Ulger, Zeynep; Kayaoglu, Guven

2014-10-01

The aim of this study was to test the dentinal tubule penetration of AH26 (Dentsply DeTrey, Konstanz, Germany) and MTA Fillapex (Angelus, Londrina, PR, Brazil) in instrumented root canals obturated by using cold lateral compaction or warm vertical compaction techniques in either the presence or absence of the smear layer. Forty-five extracted single-rooted human mandibular premolar teeth were used. The crowns were removed, and the root canals were instrumented by using the Self-Adjusting File (ReDent-Nova, Ra'anana, Israel) with continuous sodium hypochlorite (2.6%) irrigation. Final irrigation was either with 5% EDTA or with sodium hypochlorite. The canals were dried and obturated by using rhodamine B-labeled AH26 or MTA Fillapex in combination with the cold lateral compaction or the warm vertical compaction technique. After setting, the roots were sectioned horizontally at 4-, 8-, and 12-mm distances from the apical tip. On each section, sealer penetration in the dentinal tubules was measured by using confocal laser scanning microscopy. Regardless of the usage of EDTA, MTA Fillapex, compared with AH26, was associated with greater sealer penetration when used with the cold lateral compaction technique, and, conversely, AH26, compared with MTA Fillapex, was associated with greater sealer penetration when used with the warm vertical compaction technique (P < .05). Removal of the smear layer increased the penetration depth of MTA Fillapex used with the cold lateral compaction technique (P < .05); however, it had no significant effect on the penetration depth of AH26. Greater sealer penetration could be achieved with either the MTA Fillapex-cold lateral compaction combination or with the AH26-warm vertical compaction combination. Smear layer removal was critical for the penetration of MTA Fillapex; however, the same did not hold for AH26. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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...

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...

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...

High-Brightness Lasers with Spectral Beam Combining on Silicon

NASA Astrophysics Data System (ADS)

Stanton, Eric John

Modern implementations of absorption spectroscopy and infrared-countermeasures demand advanced performance and integration of high-brightness lasers, especially in the molecular fingerprint spectral region. These applications, along with others in communication, remote-sensing, and medicine, benefit from the light source comprising a multitude of frequencies. To realize this technology, a single multi-spectral optical beam of near-diffraction-limited divergence is created by combining the outputs from an array of laser sources. Full integration of such a laser is possible with direct bonding of several epitaxially-grown chips to a single silicon (Si) substrate. In this platform, an array of lasers is defined with each gain material, creating a densely spaced set of wavelengths similar to wavelength division multiplexing used in communications. Scaling the brightness of a laser typically involves increasing the active volume to produce more output power. In the direction transverse to the light propagation, larger geometries compromise the beam quality. Lengthening the cavity provides only limited scaling of the output power due to the internal losses. Individual integrated lasers have low brightness due to combination of thermal effects and high optical intensities. With heterogeneous integration, many lasers can be spectrally combined on a single integrated chip to scale brightness in a compact platform. Recent demonstrations of 2.0-microm diode and 4.8-microm quantum cascade lasers on Si have extended this heterogeneous platform beyond the telecommunications band to the mid-infrared. In this work, low-loss beam combining elements spanning the visible to the mid-infrared are developed and a high-brightness multi-spectral laser is demonstrated in the range of 4.6-4.7-microm wavelengths. An architecture is presented where light is combined in multiple stages: first within the gain-bandwidth of each laser material and then coarsely between each spectral band to a

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

PubMed

Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

2013-12-01

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Odontoma: retrospective study and confocal laser scanning microscope analysis of 52 cases.

PubMed

Crincoli, V; Scivetti, M; Di Bisceglie, M B; Lucchese, A; Favia, G

2007-01-01

The aim of this study was to perform a retrospective analysis of 52 cases of odontoma treated at the Department of Dentistry and Surgery, University of Bari, in the period 1971-2005. The odontogenic tumors were diagnosed as complex or compound odontoma following histological analysis and clinical radiological examination, and applying the 2005 WHO classification. The data analysis was conducted by considering the following factors: gender, age, site of the lesion, association with impacted teeth, aplasia, presence of supernumerary teeth as well as preoperative diagnosis by panoramic and periapical radiographs. Biopsy tissue samples were conventionally processed for histopathologic paraffin embedding and then were observed by optical microscopy and subsequently by confocal laser scanning microscopy (CLSM) in autofluorescence. Thirty specimens (57.6%) were from females and 22 (42.3%) were from males patients. The patients' age ranged from 5 to 75 years. Fifty-one percent of the specimens were excised from the mandible. In the maxilla, the most common location for odontomas was the anterior region. Most odontomas were associated with impacted teeth and only in one case there was an odontoma instead of a permanent tooth. Odontomas are considered hamartomatous malformations whose diagnosis is generally formulated by routinary radiographic examination. The CLSM analysis could help in diagnosis and histopathological analysis showing well-defined follicular area entrapped in hard tissues and pointing out ghost cells, otherwise not identifiable by traditional microscopy.

Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis.

PubMed

Skytte, Jacob L; Ghita, Ovidiu; Whelan, Paul F; Andersen, Ulf; Møller, Flemming; Dahl, Anders B; Larsen, Rasmus

2015-06-01

The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented dairy products. When studying such networks, hundreds of images can be obtained, and here image analysis methods are essential for using the images in statistical analysis. Previously, methods including gray level co-occurrence matrix analysis and fractal analysis have been used with success. However, a range of other image texture characterization methods exists. These methods describe an image by a frequency distribution of predefined image features (denoted textons). Our contribution is an investigation of the choice of image analysis methods by performing a comparative study of 7 major approaches to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis, and cluster analysis. Our investigation suggests that the texton-based descriptors provide a fuller description of the images compared to gray-level co-occurrence matrix descriptors and fractal analysis, while still being as applicable and in some cases as easy to tune. © 2015 Institute of Food Technologists®

Coherent beam combiner for a high power laser

DOEpatents

Dane, C. Brent; Hackel, Lloyd A.

2002-01-01

A phase conjugate laser mirror employing Brillouin-enhanced four wave mixing allows multiple independent laser apertures to be phase locked producing an array of diffraction-limited beams with no piston phase errors. The beam combiner has application in laser and optical systems requiring high average power, high pulse energy, and low beam divergence. A broad range of applications exist in laser systems for industrial processing, especially in the field of metal surface treatment and laser shot peening.

Coherent beam combining architectures for high power tapered laser arrays

NASA Astrophysics Data System (ADS)

Schimmel, G.; Janicot, S.; Hanna, M.; Decker, J.; Crump, P.; Erbert, G.; Witte, U.; Traub, M.; Georges, P.; Lucas-Leclin, G.

2017-02-01

Coherent beam combining (CBC) aims at increasing the spatial brightness of lasers. It consists in maintaining a constant phase relationship between different emitters, in order to combine them constructively in one single beam. We have investigated the CBC of an array of five individually-addressable high-power tapered laser diodes at λ = 976 nm, in two architectures: the first one utilizes the self-organization of the lasers in an interferometric extended-cavity, which ensures their mutual coherence; the second one relies on the injection of the emitters by a single-frequency laser diode. In both cases, the coherent combining of the phase-locked beams is ensured on the front side of the array by a transmission diffractive grating with 98% efficiency. The passive phase-locking of the laser bar is obtained up to 5 A (per emitter). An optimization algorithm is implemented to find the proper currents in the five ridge sections that ensured the maximum combined power on the front side. Under these conditions we achieve a maximum combined power of 7.5 W. In the active MOPA configuration, we can increase the currents in the tapered sections up to 6 A and get a combined power of 11.5 W, corresponding to a combining efficiency of 76%. It is limited by the beam quality of the tapered emitters and by fast phase fluctuations between emitters. Still, these results confirm the potential of CBC approaches with tapered lasers to provide a high-power and high-brightness beam, and compare with the current state-of-the-art with laser diodes.

Tracking features in retinal images of adaptive optics confocal scanning laser ophthalmoscope using KLT-SIFT algorithm

PubMed Central

Li, Hao; Lu, Jing; Shi, Guohua; Zhang, Yudong

2010-01-01

With the use of adaptive optics (AO), high-resolution microscopic imaging of living human retina in the single cell level has been achieved. In an adaptive optics confocal scanning laser ophthalmoscope (AOSLO) system, with a small field size (about 1 degree, 280 μm), the motion of the eye severely affects the stabilization of the real-time video images and results in significant distortions of the retina images. In this paper, Scale-Invariant Feature Transform (SIFT) is used to abstract stable point features from the retina images. Kanade-Lucas-Tomasi(KLT) algorithm is applied to track the features. With the tracked features, the image distortion in each frame is removed by the second-order polynomial transformation, and 10 successive frames are co-added to enhance the image quality. Features of special interest in an image can also be selected manually and tracked by KLT. A point on a cone is selected manually, and the cone is tracked from frame to frame. PMID:21258443

A statistical pixel intensity model for segmentation of confocal laser scanning microscopy images.

PubMed

Calapez, Alexandre; Rosa, Agostinho

2010-09-01

Confocal laser scanning microscopy (CLSM) has been widely used in the life sciences for the characterization of cell processes because it allows the recording of the distribution of fluorescence-tagged macromolecules on a section of the living cell. It is in fact the cornerstone of many molecular transport and interaction quantification techniques where the identification of regions of interest through image segmentation is usually a required step. In many situations, because of the complexity of the recorded cellular structures or because of the amounts of data involved, image segmentation either is too difficult or inefficient to be done by hand and automated segmentation procedures have to be considered. Given the nature of CLSM images, statistical segmentation methodologies appear as natural candidates. In this work we propose a model to be used for statistical unsupervised CLSM image segmentation. The model is derived from the CLSM image formation mechanics and its performance is compared to the existing alternatives. Results show that it provides a much better description of the data on classes characterized by their mean intensity, making it suitable not only for segmentation methodologies with known number of classes but also for use with schemes aiming at the estimation of the number of classes through the application of cluster selection criteria.

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.

Using confocal laser scanning microscopy to probe the milk fat globule membrane and associated proteins.

PubMed

Gallier, Sophie; Gragson, Derek; Jiménez-Flores, Rafael; Everett, David

2010-04-14

The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied, but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degrees of processing using two types of fluorescently labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pretreating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface.

Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

NASA Astrophysics Data System (ADS)

Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard

2012-07-01

There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

Laser diode combining for free space optical communication

NASA Technical Reports Server (NTRS)

Mecherle, G. Stephen

1986-01-01

The maximization of photon delivery to a distant collector in free space optical communications systems calls for a laser diode-combining technique employing wavelength and/or polarization as the bases of its operation. Design considerations for such a combiner encompass high throughput efficiency, diffraction-limited angular divergence, and reasonable volume constraints. Combiners are presently found to require a generalized Strehl ratio concept which includes relative source misalignment; diffraction grating combiners may have a limited number of laser sources which can meet spectral requirements. Methods for the incorporation of a combiner into a communication system are compared. Power combining is concluded to be the best tradeoff of performance and complexity for all systems, except those that are severely limited by either background radiation or component bandwidth.

Electrophoretic Detection and Confocal Microscopic Imaging of Tyrosine Nitrated Proteins in Plant Tissue.

PubMed

Arora, Dhara; Singh, Neha; Bhatla, Satish C

2018-01-01

Tyrosine nitrated proteins can be detected in plant cells electrophoretically and their distribution can be monitored by confocal laser scanning microscopy (CLSM) imaging. One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by Western blotting using polyclonal antibody against 3-nitrotyrosine residues enables detection of tyrosine nitrated proteins in plant cells. Here we describe detection of tyrosine nitrated proteins in the homogenates derived from sunflower (Helianthus annuus L.) seedling cotyledons. Total soluble proteins obtained from tissue homogenates are resolved using vertical gel electrophoresis followed by their electrophoretic transfer on to a microporous membrane support for immunodetection. Spatial distribution of tyrosine nitrated proteins can be visualized using an antibody against 3-nitrotyrosine residues. Immunofluorescent localization is performed by cutting 7 μm thick wax sections of tissue followed by incubation in primary anti-nitrotyrosine antibody (dilution 1:200) and secondary Cy-3 labeled anti-rabbit IgG antibody (dilution 1:1500). Confocal laser scanning microscopy analysis is undertaken using argon lasers (ex: 530-550 nm and em: 570 nm) at pinhole 1. Modulation in the abundance and spatial localization of tyrosine nitrated proteins in plant tissues can be monitored using these techniques.

Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

USGS Publications Warehouse

Hackley, Paul C.; Kus, Jolanta

2015-01-01

We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

Real-time mapping of the corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy

NASA Astrophysics Data System (ADS)

Guthoff, Rudolf F.; Zhivov, Andrey; Stachs, Oliver

2010-02-01

The aim of the study was to produce two-dimensional reconstruction maps of the living corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy in real time. CLSM source data (frame rate 30Hz, 384x384 pixel) were used to create large-scale maps of the scanned area by selecting the Automatic Real Time (ART) composite mode. The mapping algorithm is based on an affine transformation. Microscopy of the sub-basal nerve plexus was performed on normal and LASIK eyes as well as on rabbit eyes. Real-time mapping of the sub-basal nerve plexus was performed in large-scale up to a size of 3.2mm x 3.2mm. The developed method enables a real-time in vivo mapping of the sub-basal nerve plexus which is stringently necessary for statistically firmed conclusions about morphometric plexus alterations.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Confocal detection of Rayleigh scattering for residual stress measurement in chemically tempered glass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hödemann, S., E-mail: siim.hodemann@ut.ee; Möls, P.; Kiisk, V.

2015-12-28

A new optical method is presented for evaluation of the stress profile in chemically tempered (chemically strengthened) glass based on confocal detection of scattered laser beam. Theoretically, a lateral resolution of 0.2 μm and a depth resolution of 0.6 μm could be achieved by using a confocal microscope with high-NA immersion objective. The stress profile in the 250 μm thick surface layer of chemically tempered lithium aluminosilicate glass was measured with a high spatial resolution to illustrate the capability of the method. The confocal method is validated using transmission photoelastic and Na{sup +} ion concentration profile measurement. Compositional influence on the stress-optic coefficientmore » is calculated and discussed. Our method opens up new possibilities for three-dimensional scattered light tomography of mechanical imaging in birefringent materials.« less

In vivo three-dimensional confocal laser scanning microscopy of the epithelial nerve structure in the human cornea.

PubMed

Stachs, Oliver; Zhivov, Andrey; Kraak, Robert; Stave, Joachim; Guthoff, Rudolf

2007-04-01

Evaluation of a new method for in vivo visualization of the distribution and morphology of human anterior corneal nerves. The anterior cornea was examined to a depth of 100 microm in four human volunteers with a confocal laser scanning microscope (CLSM) using a Rostock Cornea Module (developed in house) attached to a Heidelberg Retina Tomograph II (Heidelberg Engineering, Germany). Optical sections were digitally reconstructed in 3D using AMIRA (TGS Inc., USA). The scanned volumes had a greatest size of 300 x 300 x 40 microm and voxel size of 0.78 x 0.78 x 0.95 microm. The spatial arrangement of the epithelium, nerves and keratocytes was visualized by in vivo 3D-CLSM. The 3D-reconstruction of the volunteers' corneas in combination with the oblique sections gave a picture of the nerves in the central human cornea. Thin nerves run in the subepithelial plexus aligned parallel to Bowman's layer and are partially interconnected. The diameter of these fibres varied between 1.0 and 5 microm. Thick fibres rose out of the deeper stroma. The diameter of the main nerve trunks was 12+/-2 microm. Branches penetrating the anterior epithelial cell layer could not be visualized. 3D-CLSM allows analysis of the spatial arrangement of the anterior corneal nerves and visualization of the epithelium and keratocytes in the living human cornea. The developed method provides a basis for further studies of alterations of the cellular arrangement and epithelial innervation in corneal disease. This may help to clarify alterations of nerve fibre patterns under various clinical and experimental conditions.

Classification of nanoparticle diffusion processes in vital cells by a multifeature random forests approach: application to simulated data, darkfield, and confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Wagner, Thorsten; Kroll, Alexandra; Wiemann, Martin; Lipinski, Hans-Gerd

2016-04-01

Darkfield and confocal laser scanning microscopy both allow for a simultaneous observation of live cells and single nanoparticles. Accordingly, a characterization of nanoparticle uptake and intracellular mobility appears possible within living cells. Single particle tracking makes it possible to characterize the particle and the surrounding cell. In case of free diffusion, the mean squared displacement for each trajectory of a nanoparticle can be measured which allows computing the corresponding diffusion coefficient and, if desired, converting it into the hydrodynamic diameter using the Stokes-Einstein equation and the viscosity of the fluid. However, within the more complex system of a cell's cytoplasm unrestrained diffusion is scarce and several other types of movements may occur. Thus, confined or anomalous diffusion (e.g. diffusion in porous media), active transport, and combinations thereof were described by several authors. To distinguish between these types of particle movement we developed an appropriate classification method, and simulated three types of particle motion in a 2D plane using a Monte Carlo approach: (1) normal diffusion, using random direction and step-length, (2) subdiffusion, using confinements like a reflective boundary with defined radius or reflective objects in the closer vicinity, and (3) superdiffusion, using a directed flow added to the normal diffusion. To simulate subdiffusion we devised a new method based on tracks of different length combined with equally probable obstacle interaction. Next we estimated the fractal dimension, elongation and the ratio of long-time / short-time diffusion coefficients. These features were used to train a random forests classification algorithm. The accuracy for simulated trajectories with 180 steps was 97% (95%-CI: 0.9481-0.9884). The balanced accuracy was 94%, 99% and 98% for normal-, sub- and superdiffusion, respectively. Nanoparticle tracking analysis was used with 100 nm polystyrene particles

A new diagnostic technique for tinea incognito: in vivo reflectance confocal microscopy. Report of five cases.

PubMed

Turan, Enver; Erdemir, Asli Turgut; Gurel, Mehmet Salih; Yurt, Nurdan

2013-02-01

In vivo confocal laser scanning microscopy (CLSM) is a modern non-invasive method for investigation of the skin that allows real-time visualization of individual cells and subcellular structures with the highest resolution imaging comparable to the routine histopathology. Our aim was to demonstrate the potential of CLSM for non-invasive diagnosis of difficult tinea incognito cases. Clinically atypical lesions in five cases of tinea incognito due to dermatophyte spp. were demonstrated using reflectance confocal laser scanning microscopy (RCM), parallel to KOH preparation and fungal culture of skin scrapings performed in the same patients. The morphological features characteristic for tinea incognito, namely linear branched hyphae in the intercellular area of the stratum corneum, were readily detectable by means of CLSM. In vivo tissue imaging were performed at three different wavelengths (785, 658, 445 nm) and the best images of fungal elements were obtained at 445 nm. All of our five cases had similar reflectance confocal microscopical findings. Our findings suggest the potential of CLSM as a non-invasive tool for the diagnosis of tinea incognito having atypical clinical appearance. Although at present the reflectance confocal microscopy cannot replace the current diagnostic standards for tinea incognito, it may be successfully used as in vivo non-invasive screening tool to facilitate the diagnosis and point to the need for further investigation of the patient. © 2012 John Wiley & Sons A/S.

Fabry-Perot confocal resonator optical associative memory

NASA Astrophysics Data System (ADS)

Burns, Thomas J.; Rogers, Steven K.; Vogel, George A.

1993-03-01

A unique optical associative memory architecture is presented that combines the optical processing environment of a Fabry-Perot confocal resonator with the dynamic storage and recall properties of volume holograms. The confocal resonator reduces the size and complexity of previous associative memory architectures by folding a large number of discrete optical components into an integrated, compact optical processing environment. Experimental results demonstrate the system is capable of recalling a complete object from memory when presented with partial information about the object. A Fourier optics model of the system's operation shows it implements a spatially continuous version of a discrete, binary Hopfield neural network associative memory.

Effect of laser power on clad metal in laser-TIG combined metal cladding

NASA Astrophysics Data System (ADS)

Utsumi, Akihiro; Hino, Takanori; Matsuda, Jun; Tasoda, Takashi; Yoneda, Masafumi; Katsumura, Munehide; Yano, Tetsuo; Araki, Takao

2003-03-01

TIG arc welding has been used to date as a method for clad welding of white metal as bearing material. We propose a new clad welding process that combines a CO2 laser and a TIG arc, as a method for cladding at high speed. We hypothesized that this method would permit appropriate control of the melted quantity of base metal by varying the laser power. We carried out cladding while varying the laser power, and investigated the structure near the boundary between the clad layer and the base metal. Using the laser-TIG combined cladding, we found we were able to control appropriately the degree of dilution with the base metal. By applying this result to subsequent cladding, we were able to obtain a clad layer of high quality, which was slightly diluted with the base metal.

Feasibility of confocal endomicroscopy in the diagnosis of pediatric gastrointestinal disorders

PubMed Central

Venkatesh, Krishnappa; Cohen, Marta; Evans, Clair; Delaney, Peter; Thomas, Steven; Taylor, Christopher; Abou-Taleb, Ashraf; Kiesslich, Ralf; Thomson, Mike

2009-01-01

AIM: To evaluate the feasibility and utility of confocal laser endomicroscopy (CLE) in the description of normal gastrointestinal (GI) mucosa and in the diagnosis of GI disorders in children, in comparison to histology. METHODS: Forty-four patients (19 female) median age 10.9 years (range 0.7-16.6 years) with suspected or known GI pathology underwent esophago-gastro-duodenoscopy (OGD) (n = 36) and/or ileocolonoscopy (IC) (n = 31) with CLE using sodium fluorescein and acriflavine as contrast agents. Histological sections were compared with same site confocal images by two experienced pediatric and GI histopathologists and endoscopists, respectively. RESULTS: Duodenum and ileum were intubated in all but one patient undergoing OGD and IC. The median procedure time was 16.4 min (range 7-25 min) for OGD and 27.9 min (range 15-45 min) for IC. A total of 4798 confocal images were compared with 153 biopsies from the upper GI tract from 36 procedures, and 4661 confocal images were compared with 188 biopsies from the ileocolon from 31 procedures. Confocal images were comparable to conventional histology both in normal and in pathological conditions such as esophagitis, Helicobacter pylori gastritis, celiac disease, inflammatory bowel disease, colonic heterotopia, and graft versus host disease. CONCLUSION: CLE offers the prospect of targeting biopsies to abnormal mucosa, thereby increasing diagnostic yield, reducing the number of biopsies, decreasing the burden on the histopathological services, and reducing costs. PMID:19437560

In-situ investigation of thermal instabilities and solid state dewetting in polycrystalline platinum thin films via confocal laser microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jahangir, S.; Cheng, Xuan; Huang, H. H.

2014-10-28

Solid state dewetting and the subsequent morphological changes for platinum thin films grown on zinc oxide (ZnO) buffered (001) silicon substrates (Pt/ZnO/SiO{sub 2}/(001)Si system) is investigated under vacuum conditions via a custom-designed confocal laser microscope coupled with a laser heating system. Live imaging of thin film dewetting under a range of heating and quenching vacuum ambients reveals events including hillock formation, hole formation, and hole growth that lead to formation of a network of Pt ligaments, break up of Pt ligaments to individual islands and subsequent Pt islands shape reformation, in chronological fashion. These findings are corroborated by ex-situ materialsmore » characterization and quantitative electron microscopy analysis. A secondary hole formation via blistering before film rupture is revealed to be the critical stage, after which a rapid dewetting catastrophe occurs. This process is instantaneous and cannot be captured by ex-situ methods. Finally, an intermetallic phase forms at 900 °C and alters the morphology of Pt islands, suggesting a practical limit to the thermal environments that may be used for these platinized silicon wafers in vacuum conditions.« less

Comparison of pulsed dye laser versus combined pulsed dye laser and Nd:YAG laser in the treatment of inflammatory acne vulgaris.

PubMed

Salah El Din, Manal Mohamed; Samy, Nevien Ahmed; Salem, Amira Eid

2017-06-01

Both pulsed dye laser and combined 585/1064-nm (sequential dual-wavelength PDL and Nd:YAG) laser improves inflammatory skin disorders including acne vulgaris. To compare the efficacy of 585-nm pulsed dye laser versus sequential dual-wavelength PDL and Nd:YAG in treatment of acne vulgaris. Thirty patients with acne vulgaris were treated by PDL alone on half of the face while contra lateral half was treated by combined 585/1064 nm laser. The study showed that inflammatory acne lesions count was significantly reduced by 82.5% (p 0.0001) on PDL sides and by 83.5% (p 0.00001) on combined 585/1064-nm side after 8 weeks, while reduction of non-inflammatory acne lesions was observed at 8 weeks by 58.4% and 71.5% respectively. However, difference between the two modalities was not statistically significant. PDL and combined PDL/Nd:YAG laser treatment were found to be an effective, safe and well-tolerated treatment option for inflammatory and non-inflammatory acne vulgaris.

Combining immunolabeling and catalyzed reporter deposition to detect intracellular saxitoxin in a cyanobacterium.

PubMed

Piccini, Claudia; Fabre, Amelia; Lacerot, Gissell; Bonilla, Sylvia

2015-10-01

We combined the use of polyclonal antibodies against saxitoxin with catalyzed reporter deposition to detect production of saxitoxin by the cyanobacterium Cylindrospermopsis raciborskii. The procedure is simple, allows detection of intracellular saxitoxin in cyanobacteria filaments by confocal laser microscopy and is a promising tool to study toxin production and metabolism. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of a High Spectral Resolution Lidar (HSRL) Based on a Confocal Optical Filter for Aerosol Studies

NASA Astrophysics Data System (ADS)

Repasky, K. S.; Hoffman, D. S.; Reagan, J. A.; Carlsten, J.

2010-12-01

Aerosols are an important constituent in atmospheric composition affecting climate, weather, and air quality. Active remote sensing instruments provide tools for in-situ studies of atmospheric aerosols that can help understand the role of aerosols on the radiative forcing of the climate system. In this paper, the design and initial performance of a high spectral resolution lidar (HSRL) based on a unique confocal cavity for optically filtering the aerosol and molecular returns is presented. An injection seeded pulsed Nd:YAG laser with a fundamental and frequency doubled output is used as the laser transmitter for the HSRL. A small portion of fiber coupled injection seeded signal at 1064 nm is split before the laser oscillator and, after modulation using an acousto-optic modulator, is used to produce a discriminating signal for locking a confocal cavity that is resonant at the 1064 and 532 nm wavelengths to the injection seeded source. Light scattered in the atmosphere is collected using a commercial telescope. After the telescope, the 1064 nm light is split from the 532 nm light using a dielectric mirror with the 1064 nm light monitored using a PMT. The 532 nm light is launched into a multimode fiber. The output from the fiber is next incident on a beamsplitter with part of the light sent to a PMT to monitor the total return for the 532 nm channel. The light that passes through the beamsplitter is mode matched into a confocal optical cavity that allows the light scattered by the atmospheric aerosols to be transmitted while the light scattered from the atmospheric molecules is reflected. The transmitted light from the aerosol scattering is incident on a PMT while the reflected molecular signal is incident on a PMT. The transmission of the confocal cavity is monitored before and after the data collection using a continuous wave frequency doubled Nd:YAG laser that is fiber coupled. Data is collected and processed in the following manner. Each of the four voltage

Invasive leg vein treatment with 1064/1319 Nd:YAG laser: combination with dye laser treatment

NASA Astrophysics Data System (ADS)

Smucler, Roman; Horak, Ladislav; Mazanek, Jiri

1999-06-01

More than 2 500 leg veins patients were treated with dye laser / ScleroPlus, Candela, USA / successfully in our clinic and we use this therapy as the basic cosmetics treatment. But especially diameter of leg vein is limiting factor. Very often we have to treat some cases that are not ideal for classical surgical or for dye laser method. We decided to make invasive perivenous laser coagulation. We adapted original Czech 1064/1319 nm Nd:YAG laser / US patent pending /, which is new combine tool, for invasive application. Principe: After we have penetrated the cutis with laser fiber we coagulate leg veins during slowly perivenous motion. Perfect preoperative examination is a condition of success. After 15 months we have very interesting results. Some patients / 15%/ were perfect treated only with this possibility but excellent results are acquired from combination with dye laser.

Spatial Combining of Laser-Diode Beams for Pumping an NPRO

NASA Technical Reports Server (NTRS)

Gelsinger, Paul; Liu, Duncan; Mulder, Jerry; Aguayo, Francisco

2008-01-01

A free-space optical beam combiner now undergoing development makes it possible to use the outputs of multiple multimode laser diodes to pump a neodymium-doped yttrium aluminum garnet (Nd:YAG) non-planar ring oscillator (NPRO) laser while ensuring that the laser operates at only a single desired frequency. Heretofore, a Nd:YAG NPRO like the present one has been pumped by a single multimode laser-diode beam delivered via an optical fiber. It would be desirable to use multiple pump laser diodes to increase reliability beyond that obtainable from a single pump laser diode. However, as explained in this article, simplistically coupling multiple multimode laser-diode beams through a fiber-optic combiner would entail a significant reduction in coupling efficiency, and lasing would occur at one or more other frequencies in addition to the single desired frequency. To minimize coupling loss, one must ensure that the NA (approximately equal to 0.3) of the combined laser-diode beams is less than the NA of the fiber. The A(Omega) of the laser-diode beam in the slow-axis plane is 1/1.3 as large as that of the fiber. This A(Omega) is small enough to enable efficient coupling of light into the optical fiber, but too large for combining of beams in the slow-axis plane. Therefore, a pair of cylindrical lenses is used to cancel the slow-axis plane magnification introduced by the on-cylindrical lenses used to effect magnification in the fast-axis plane.

Diffractive Combiner of Single-Mode Pump Laser-Diode Beams

NASA Technical Reports Server (NTRS)

Liu, Duncan; Wilson, Daniel; Qiu, Yueming; Forouhar, Siamak

2007-01-01

An optical beam combiner now under development would make it possible to use the outputs of multiple single-mode laser diodes to pump a neodymium: yttrium aluminum garnet (Nd:YAG) nonplanar ring oscillator (NPRO) laser while ensuring that the laser operates at only a single desired frequency. Heretofore, an Nd:YAG NPRO like the present one has been pumped by a single multimode laser-diode beam delivered via an optical fiber. It would be desirable to use multiple pump laser diodes to increase reliability beyond that obtainable from a single pump laser diode. However, as explained below, simplistically coupling multiple multimode laser-diode beams through a fiber-optic combiner would entail a significant reduction in coupling efficiency, and lasing would occur at one or more other frequencies in addition to the single desired frequency. Figure 1 schematically illustrates the principle of operation of a laser-diode-pumped Nd:YAG NPRO. The laser beam path is confined in a Nd:YAG crystal by means of total internal reflections on the three back facets and a partial-reflection coating on the front facet. The wavelength of the pump beam - 808 nm - is the wavelength most strongly absorbed by the Nd:YAG crystal. The crystal can lase at a wavelength of either 1,064 nm or 1,319 nm - which one depending on the optical coating on the front facet. A thermal lens effect induced by the pump beam enables stable lasing in the lowest-order transverse electromagnetic mode (the TEM00 mode). The frequency of this laser is very stable because of the mechanical stability of the laser crystal and the unidirectional nature of the lasing. The unidirectionality is a result of the combined effects of (1) a Faraday rotation induced by an externally applied magnetic field and (2) polarization associated with non-normal incidence and reflection on the front facet.

Fused oblique incidence reflectometry and confocal fluorescence microscopy

NASA Astrophysics Data System (ADS)

Risi, Matthew D.; Rouse, Andrew R.; Gmitro, Arthur F.

2011-03-01

Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure, but relies on exogenous fluorophores, has a relatively limited penetration depth (100 μm) and field of view (700 μm), and produces a high rate of detailed information to the user. A new catheter based multi-modal system has been designed that combines confocal imaging and oblique incidence reflectometry (OIR), which is a non-invasive method capable of rapidly extracting tissue absorption, μa, and reduced scattering, μ's, spectra from tissue. The system builds on previous developments of a custom slit-scan multi-spectral confocal microendoscope and is designed to rapidly switch between diffuse spectroscopy and confocal fluorescence imaging modes of operation. An experimental proof-of-principle catheter has been developed that consists of a fiber bundle for traditional confocal fluorescence imaging and a single OIR source fiber which is manually redirected at +/- 26 degrees. Diffusely scattered light from each orientation of the source fiber is collected via the fiber bundle, with a frame of data representing spectra collected at a range of distances from the OIR source point. Initial results with intralipid phantoms show good agreement to published data over the 550-650 nm spectral range. We successfully imaged and measured the optical properties of rodent cardiac muscle.

In vivo confocal microscopy, an inner vision of the cornea - a major review.

PubMed

Guthoff, Rudolf F; Zhivov, Andrey; Stachs, Oliver

2009-01-01

The demands of modern ophthalmology have evolved from descriptive findings from the slit lamp to in vivo assessment of cellular level changes. Nowadays, the latter can be provided by in vivo confocal microscopy. This article gives an overview of confocal principles using tandem scanning, scanning slit and laser scanning techniques used in ophthalmology. The main part of the paper describes the clinical applications emphasizing the anatomy of the normal and pathological cornea, and illustrates side-effects of topical medication, contact lens wear, cross-linking and refractive surgery. Finally, a summary about experimental applications, including animal studies, surface characterization and volume rendering as well as future developments, is given.

Correlative imaging of biological tissues with apertureless scanning near-field optical microscopy and confocal laser scanning microscopy

PubMed Central

Stanciu, Stefan G.; Tranca, Denis E.; Hristu, Radu; Stanciu, George A.

2017-01-01

Apertureless scanning near-field optical microscopy (ASNOM) has attracted considerable interest over the past years as a result of its valuable contrast mechanisms and capabilities for optical resolutions in the nanoscale range. However, at this moment the intersections between ASNOM and the realm of bioimaging are scarce, mainly due to data interpretation difficulties linked to the limited body of work performed so far in this field and hence the reduced volume of supporting information. We propose an imaging approach that holds significant potential for alleviating this issue, consisting of correlative imaging of biological specimens using a multimodal system that incorporates ASNOM and confocal laser scanning microscopy (CLSM), which allows placing near-field data into a well understood context of anatomical relevance. We demonstrate this approach on zebrafish retinal tissue. The proposed method holds important implications for the in-depth understanding of biological items through the prism of ASNOM and CLSM data complementarity. PMID:29296474

Application of reflectance confocal microscopy to evaluate skin damage after irradiation with an yttrium-scandium-gallium-garnet (YSGG) laser.

PubMed

Yue, Xueping; Wang, Hongwei; Li, Qing; Li, Linfeng

2017-02-01

The objective of this study was to observe the characteristics of the skin after irradiation with a 2790-nm yttrium-scandium-gallium-garnet (YSGG) laser using reflectance confocal microscopy (RCM). A 2790-nm YSGG laser was used to irradiate fresh foreskin (four doses, at spot density 3) in vitro. The characteristics of microscopic ablative columns (MAC), thermal coagulation zone (TCZ), and microscopic treatment zones (MTZ) were observed immediately after irradiation using digital microscope and RCM. The characteristics of MAC, TCZ, and MTZ with variations in pulse energy were comparatively analyzed. After irradiation, MAC, TCZ, and MTZ characteristics and undamaged skin between MTZs can be observed by RCM. The depth and width of MTZ obviously increased with the increase in pulse energy. At 80, 120, and 160 mJ/microbeam (MB), the MTZ actual area and proportion were about two times that of the theoretical value and three times at 200 mJ/MB. With increases in depth, the single MAC gradually decreased in a fingertip-shaped model, with TCZ slowly increasing, and MTZ slightly decreasing in a columnar shape. RCM was able to determine the characteristics of thermal injury on the skin after the 2790-nm YSGG laser irradiation with different pulse energies. Pulse energy higher than 200 mJ/MB may have much larger thermal injury and side effect. RCM could be used in the clinic in future.

Nano material processing with lasers in combination with nearfield technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dickmann, K.; Jersch, J.; Demming, F.

1996-12-31

Recent research work has shown, that focusing of laser radiation down to a few nanometer can be obtained by using lasers in combination with nearfield technology (known from Scanning Tunneling Microscope STM or Atomic Force Microscope AFM). Lateral external illumination of STM- or AFM-probe tips with laser radiation can cause tremendous intensity enhancement in the nearfield underneath the tip. This effect can be explained by various electrostatic as well as electrodynamic effects known from Surface Enhanced Raman Spectroscopy (SERS). This effect was utilized to concentrate laser radiation with high intensity between a tip and a substrate in the nearfield. FOLANT-techniquemore » (FOcusing of LAser radiation in the Nearfield of a Tip) enables intensity enhancement up to 10{sup 6} in a narrow localized zone underneath the tip. The interaction area with nanometer scale can be applied for material processing even down to atomic dimensions. Using STM-/ laser-combination, hillocks, pits and grooves with lateral dimensions down to 10 nm have been obtained on gold substrates. AFM-/ laser-combination enabled nanostructures down to 20 nm on dielectric materials as for example polycarbonate.« less

Fusion welding studies using laser on Ti-SS dissimilar combination

NASA Astrophysics Data System (ADS)

Shanmugarajan, B.; Padmanabham, G.

2012-11-01

Laser welding investigations were carried out on dissimilar Ti-SS combination. The study is aimed to improve the weld strength and ductility by minimizing harmful intermetallics and taking advantage of high cooling rates in laser welding. Results of continuous wave 3.5 kW CO2 laser welding of totally dissimilar combination of Titanium and stainless steel (304) have been discussed. Bead on plate welding experiments were conducted to identify the laser welding parameters using depth of penetration as criteria. The welding of dissimilar combination has been attempted both autogenously and with interlayers such as Vanadium (V) and Tantalum (Ta) in the form of laser cladding as well as strip. Autogenous welds were carried out by varying the laser power, welding speed and position of the laser beam with respect to the joint centre. The resultant welds are characterized by macrostructure analysis, SEM/EDAX and XRD and as welded tensile test in UTM. The autogenous welds have exhibited extensive cracking even when welded at high speeds or by manipulating the beam position with respect to the joint. Similarly Vandaium as interlayer could not achieve crack free joint. A joint with 40 MPa strength could be made with Ta as interlayer. Results and analysis of these variants of laser welded joints are reported and discussed.

Hypericin and pulsed laser therapy of squamous cell cancer in vitro.

PubMed

Bublik, Michael; Head, Christian; Benharash, Peyman; Paiva, Marcos; Eshraghi, Adrian; Kim, Taiho; Saxton, Romaine

2006-06-01

This in vitro study compares continuous wave and pulsed laser light at longer wavelengths for activation of the phototoxic drug hypericin in human cancer cells. Two-photon pulsed laser light now allows high-resolution fluorescent imaging of cancer cells and should provide deeper tissue penetration with near infrared light for improved detection as well as phototoxicity in human tumors. Cultured Seoul National University (SNU)-1 tumor cells from a squamous cell carcinoma (SCC) were incubated with hypericin before photoirradiation at four laser wavelengths. Phototoxicity of hypericin sensitized SCC cells was measured by dimethyl thiazoldiphenyl (MTT) tetrazolium bromide cell viability assays and by confocal fluorescence microscopy via 532-nm and infrared two-photon pulsed laser light. Phototoxic response increased linearly with hypericin dose of 0.1-2 microM, light exposure time of 5-120 sec, and pulsed dye laser wavelengths of 514-593 nm. Light energy delivery for 50% cell phototoxicity (LD50) response was 9 joules at 514 nm, 3 joules at 550 nm, and less than 1 joule at the 593 nm hypericin light absorption maxima. Fluorescence confocal microscopy revealed membrane and perinuclear localization of hypericin in the SNU cells with membrane damage seen after excitation with visible 532 nm continuous wave light or two-photon 700-950 nm picosecond pulsed laser irradiation. Hypericin may be a powerful tumor targetting drug when combined with pulsed laser light in patients with recurrent head and neck SCC.

An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography.

PubMed

Mochalov, Konstantin E; Chistyakov, Anton A; Solovyeva, Daria O; Mezin, Alexey V; Oleinikov, Vladimir A; Vaskan, Ivan S; Molinari, Michael; Agapov, Igor I; Nabiev, Igor; Efimov, Anton E

2017-11-01

In the past decade correlative microscopy, which combines the potentials of different types of high-resolution microscopies with a variety of optical microspectroscopy techniques, has been attracting increasing attention in material science and biological research. One of outstanding solutions in this area is the combination of scanning probe microscopy (SPM), which provides data on not only the topography, but also the spatial distribution of a wide range of physical properties (elasticity, conductivity, etc.), with ultramicrotomy, allowing 3D multiparametric examination of materials. The combination of SPM and ultramicrotomy (scanning probe nanotomography) is very appropriate for characterization of soft multicompound nanostructurized materials, such as polymer matrices and microstructures doped with different types of nanoparticles (magnetic nanoparticles, quantum dots, nanotubes, etc.), and biological materials. A serious problem of this technique is a lack of chemical and optical characterization tools, which may be solved by using optical microspectroscopy. Here, we report the development of an instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography in a single apparatus. This approach retains all the advantages of SPM and upright optical microspectroscopy and allows 3D multiparametric characterization using both techniques. As the first test of the system developed, we have performed correlative characterization of the morphology and the magnetic and fluorescent properties of fluorescent magnetic microspheres doped with a fluorescent dye and magnetic nanoparticles. The results of this study can be used to obtain 3D volume images of a specimen for most high-resolution near-field scanning probe microscopies: SNOM, TERS, AFM-IR, etc. This approach will result in development of unique techniques combining the advantages of SPM (nanoscale morphology and a wide range of physical parameters) and high-resolution optical

Multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy for image-guided feedback of intraocular injections in mouse models

NASA Astrophysics Data System (ADS)

Benavides, Oscar R.; Terrones, Benjamin D.; Leeburg, Kelsey C.; Mehanathan, Sankarathi B.; Levine, Edward M.; Tao, Yuankai K.

2018-02-01

Rodent models are robust tools for understanding human retinal disease and function because of their similarities with human physiology and anatomy and availability of genetic mutants. Optical coherence tomography (OCT) has been well-established for ophthalmic imaging in rodents and enables depth-resolved visualization of structures and image-based surrogate biomarkers of disease. Similarly, fluorescence confocal scanning laser ophthalmoscopy (cSLO) has demonstrated utility for imaging endogenous and exogenous fluorescence and scattering contrast in the mouse retina. Complementary volumetric scattering and en face fluorescence contrast from OCT and cSLO, respectively, enables cellular-resolution longitudinal imaging of changes in ophthalmic structure and function. We present a non-contact multimodal OCT+cSLO small animal imaging system with extended working distance to the pupil, which enables imaging during and after intraocular injection. While injections are routinely performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the location and volume delivered is not precisely controlled and difficult to reproduce. Animals were imaged using a custom-built OCT engine and scan-head combined with a modified commercial cSLO scan-head. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. When combined with imagesegmentation, we believe OCT can be used to precisely identify injection locations and quantify injection volumes. Fluorescence cSLO can provide complementary contrast for either fluorescently labeled compounds or transgenic cells for improved specificity. Our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections, which may be used for real-time image-guided injections.

In vivo laser confocal microscopy of Bowman's layer of the cornea.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2006-12-01

To investigate in vivo microstructures of Bowman's layer in normal human subjects using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT2-RCM). Single-center, prospective, observational case series. Nineteen normal volunteers (10 male, 9 female; mean age, 46.2+/-21.7 years [range, 18-77]). The central and peripheral cornea, specifically the epithelium, Bowman's layer, and its subjacent stroma, were examined using the HRT2-RCM. Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the microstructures. In all subjects, normal epithelial (superficial, wing, basal) cells, subbasal nerve plexus, Bowman's layer, and its subjacent stoma were observed clearly. However, in all subjects, polymorphic structures composed of fibrillar materials with less reflectivity than corneal nerves were observed beneath Bowman's layer. After application of pressure by a Tomo-cap, we observed numerous ridges that protruded into the epithelial basal and wing cell layers. Superficial stromal striae were also observed. These ridges and striae corresponded exactly to the orientation of the fibrous structures located beneath the epithelial cells. We report for the first time, the presence of polymorphic structures composed of fibrillar materials (K-structures) beneath Bowman's layer in normal human subjects, detected by HRT2-RCM. We surmise that these microstructures may correspond to the modified and condensed anterior stromal collagen fibers/lamellae that merge into Bowman's layer and that these fibrillar materials may be responsible for the formation of the anterior corneal mosaic. Further investigation of these microstructures in diseased eyes may provide insights into their pathophysiologic role in Bowman's layer.

Numerical simulation of dendrite growth in nickel-based superalloy and validated by in-situ observation using high temperature confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Yan, Xuewei; Xu, Qingyan; Liu, Baicheng

2017-12-01

Dendritic structures are the predominant microstructural constituents of nickel-based superalloys, an understanding of the dendrite growth is required in order to obtain the desirable microstructure and improve the performance of castings. For this reason, numerical simulation method and an in-situ observation technology by employing high temperature confocal laser scanning microscopy (HT-CLSM) were used to investigate dendrite growth during solidification process. A combined cellular automaton-finite difference (CA-FD) model allowing for the prediction of dendrite growth of binary alloys was developed. The algorithm of cells capture was modified, and a deterministic cellular automaton (DCA) model was proposed to describe neighborhood tracking. The dendrite and detail morphology, especially hundreds of dendrites distribution at a large scale and three-dimensional (3-D) polycrystalline growth, were successfully simulated based on this model. The dendritic morphologies of samples before and after HT-CLSM were both observed by optical microscope (OM) and scanning electron microscope (SEM). The experimental observations presented a reasonable agreement with the simulation results. It was also found that primary or secondary dendrite arm spacing, and segregation pattern were significantly influenced by dendrite growth. Furthermore, the directional solidification (DS) dendritic evolution behavior and detail morphology were also simulated based on the proposed model, and the simulation results also agree well with experimental results.

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...

Real-Time Demonstration of Split Skin Graft Inosculation and Integra Dermal Matrix Neovascularization Using Confocal Laser Scanning Microscopy

PubMed Central

Greenwood, John; Amjadi, Mahyar; Dearman, Bronwyn; Mackie, Ian

2009-01-01

Objectives: During the first 48 hours after placement, an autograft “drinks” nutrients and dissolved oxygen from fluid exuding from the underlying recipient bed (“plasmatic imbibition”). The theory of inosculation (that skin grafts subsequently obtain nourishment via blood vessel “anastomosis” between new vessels invading from the wound bed and existing graft vessels) was hotly debated from the late 19th to mid-20th century. This study aimed to noninvasively observe blood flow in split skin grafts and Integra™ dermal regeneration matrix to provide further proof of inosculation and to contrast the structure of vascularization in both materials, reflecting mechanism. Methods: Observations were made both clinically and using confocal microscopy on normal skin, split skin graft, and Integra™. The VivaScope™ allows noninvasive, real-time, in vivo images of tissue to be obtained. Results: Observations of blood flow and tissue architecture in autologous skin graft and Integra™ suggest that 2 very different processes are occurring in the establishment of circulation in each case. Inosculation provides rapid circulatory return to skin grafts whereas slower neovascularization creates an unusual initial Integra™ circulation. Conclusions: The advent of confocal laser microscopy like the VivaScope 1500™, together with “virtual” journals such as ePlasty, enables us to provide exciting images and distribute them widely to a “reading” audience. The development of the early Integra™ vasculature by neovascularization results in a large-vessel, high-volume, rapid flow circulation contrasting markedly from the inosculatory process in skin grafts and the capillary circulation in normal skin and merits further (planned) investigation. PMID:19787028

Fluorescence fibre-optic confocal microscopy of skin in vivo: microscope and fluorophores.

PubMed

Suihko, Christian; Swindle, Lucinda D; Thomas, Steven G; Serup, Jørgen

2005-11-01

Fibre-optic confocal imaging in vivo is a new approach in the assessment of human skin. The objective is to describe a novel instrument and its operation and use in combination with fluorophores. The Stratum is a fibre-optic fluorescence confocal microscope especially developed for the study of skin and mucous membranes. The system is flexible and any body site can be studied with a hand-held scanner. The light source is a 488 nm argon ion laser. Horizontal (en face) images of the epidermis and outer dermis are produced with cellular resolution. Magnification is approximately 1000 x . Fluorescein sodium is routinely used as fluorophore (intradermal injection or application to the skin surface). This fluorophore is safe for human use in vivo, but other substances (rhodamine B, Acridine Orange, green fluorescent protein, curcumin) have also been studied. The instrument produces sharp images of epidermal cell layers from the epidermal surface to the sub-papillary dermis, with sub-cellular resolution. The scanner is flexible in use. The technique of intradermal fluorophore injection requires some skill. We consider this fibre-optic instrument a potentially important tool in skin research for non-invasive optical biopsy of primarily the epidermis. Present use is focussed on research applications, where the fluorophore distribution in the skin may illustrate morphological changes in the epidermis.

Confocal Microscopy Imaging with an Optical Transition Edge Sensor

NASA Astrophysics Data System (ADS)

Fukuda, D.; Niwa, K.; Hattori, K.; Inoue, S.; Kobayashi, R.; Numata, T.

2018-05-01

Fluorescence color imaging at an extremely low excitation intensity was performed using an optical transition edge sensor (TES) embedded in a confocal microscope for the first time. Optical TES has the ability to resolve incident single photon energy; therefore, the wavelength of each photon can be measured without spectroscopic elements such as diffraction gratings. As target objects, animal cells labeled with two fluorescent dyes were irradiated with an excitation laser at an intensity below 1 μW. In our confocal system, an optical fiber-coupled TES device is used to detect photons instead of the pinhole and photomultiplier tube used in typical confocal microscopes. Photons emitted from the dyes were collected by the objective lens, and sent to the optical TES via the fiber. The TES measures the wavelength of each photon arriving in an exposure time of 70 ms, and a fluorescent photon spectrum is constructed. This measurement is repeated by scanning the target sample, and finally a two-dimensional RGB-color image is obtained. The obtained image showed that the photons emitted from the dyes of mitochondria and cytoskeletons were clearly resolved at a detection intensity level of tens of photons. TES exhibits ideal performance as a photon detector with a low dark count rate (< 1 Hz) and wavelength resolving power. In the single-mode fiber-coupled system, the confocal microscope can be operated in the super-resolution mode. These features are very promising to realize high-sensitivity and high-resolution photon spectral imaging, and would help avoid cell damage and photobleaching of fluorescence dyes.

Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

PubMed Central

Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

2016-01-01

Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

PubMed

Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

2016-01-01

Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.

PubMed

Merdan, Thomas; Kunath, Klaus; Fischer, Dagmar; Kopecek, Jindrich; Kissel, Thomas

2002-02-01

Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.

Fluorescence confocal microscopy for pathologists.

PubMed

Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

Mapping of Heavy Metal Ion Sorption to Cell-Extracellular Polymeric Substance-Mineral Aggregates by Using Metal-Selective Fluorescent Probes and Confocal Laser Scanning Microscopy

PubMed Central

Li, Jianli; Kappler, Andreas; Obst, Martin

2013-01-01

Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe3+, Cu2+, Zn2+, and Hg2+, illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems. PMID:23974141

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

PubMed

Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

2015-02-07

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.

Confocal bioimaging the living cornea with autofluorescence and specific fluorescent probes

NASA Astrophysics Data System (ADS)

Masters, Barry R.; Paddock, Stephen W.

1990-08-01

Confocal bioimaging of the fine structure of the living rabbit cornea with both reflected light and fluorescent light has been demonstrated with a laser scanning confocal imaging system. Kalman averaging was used to reduce the noise in the images. Superficial epithelial, basal epithelial cells, stromal keratocytes, and endothelial cells were imaged. These cells and their subcellular structures were imaged in the two modes for comparison. The superficial epithelial cells were imaged by their autofluorescence (488/520 nm). This fluorescence signal may be due to the mitochondrial flavoproteins and can be used as a noninvasive indicator of cellular oxidative function. Thiazole orange was used to stain cell nuclei for fluorescence imaging. DiOC6 was used to stain the endoplasmic reticulum for fluorescence imaging. Fluorescein- conjugated phalloidin was used to stain actin for fluorescence imaging.

Combining Raman and laser induced breakdown spectroscopy by double pulse lasing.

PubMed

Lednev, Vasily N; Pershin, Sergey M; Sdvizhenskii, Pavel A; Grishin, Mikhail Ya; Fedorov, Alexander N; Bukin, Vladimir V; Oshurko, Vadim B; Shchegolikhin, Alexander N

2018-01-01

A new approach combining Raman spectrometry and laser induced breakdown spectrometry (LIBS) within a single laser event was suggested. A pulsed solid state Nd:YAG laser running in double pulse mode (two frequency-doubled sequential nanosecond laser pulses with dozens microseconds delay) was used to combine two spectrometry methods within a single instrument (Raman/LIBS spectrometer). First, a low-energy laser pulse (power density far below ablation threshold) was used for Raman measurements while a second powerful laser pulse created the plasma suitable for LIBS analysis. A short time delay between two successive pulses allows measuring LIBS and Raman spectra at different moments but within a single laser flash-lamp pumping. Principal advantages of the developed instrument include high quality Raman/LIBS spectra acquisition (due to optimal gating for Raman/LIBS independently) and absence of target thermal alteration during Raman measurements. A series of high quality Raman and LIBS spectra were acquired for inorganic salts (gypsum, anhydrite) as well as for pharmaceutical samples (acetylsalicylic acid). To the best of our knowledge, the quantitative analysis feasibility by combined Raman/LIBS instrument was demonstrated for the first time by calibration curves construction for acetylsalicylic acid (Raman) and copper (LIBS) in gypsum matrix. Combining ablation pulses and Raman measurements (LIBS/Raman measurements) within a single instrument makes it an efficient tool for identification of samples hidden by non-transparent covering or performing depth profiling analysis including remote sensing. Graphical abstract Combining Raman and laser induced breakdown spectroscopy by double pulse lasing.

Design considerations of a real-time clinical confocal microscope

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-06-01

A real-time clinical confocal light microscope provides the ophthalmologist with a new tool for the observation of the cornea and the ocular lens. In addition, the ciliary body, the iris, and the sclera can be observed. The real-time light microscopic images have high contrast and resolution. The transverse resolution is about one half micron and the range resolution is one micron. The following observations were made with visible light: corneal epithelial cells, wing cells, basal cells, Bowman's membrane, nerve fibers, basal lamina, fibroblast nuclei, Descemet's membrane, endothelial cells. Observation of the in situ ocular lens showed lens capsule, lens epithelium, lens fibrils, the interior of lens fibrils. The applications of the confocal microscope include: eye banking, laser refractive surgery, observation of wound healing, observation of the iris, the sciera, the ciliary body, the ocular lens, and the intraocular lens. Digital image processing can produce three-dimensional reconstructions of the cornea and the ocular lens.

Noninvasive detection and staging of oral cancer in vivo with confocal optoacoustic tomography

NASA Astrophysics Data System (ADS)

Savateeva, Elena V.; Karabutov, Alexander A.; Motamedi, Massoud; Bell, Brent A.; Johnigan, Richard M.; Oraevsky, Alexander A.

2000-05-01

Confocal opto-acoustic transducer (COAT) was developed and applied for detection of early stages of squamous cell carcinoma in hamster model of oral cancer. COAT is a novel imaging modality with optical and acoustic lens utilized for detecting in-depth opto-acoustic front surface transducer is an improved lateral resolution of 60-micrometers . The bandwidth of the confocal opto-acoustic transducer is more than 100 MHz. Therefore, in-depth axial resolution defined by the laser pulse duration and detection system equals 15-micrometers . Imaging was performed at the wavelength of the Nd:YAG laser second harmonic, which provided sufficient depth of monitoring and significant tissue contrast. Correlation of the opto- acoustic images with H and E histology sections in control animals and in animals treated with carcinogenic agent, DMBA, confirmed previous findings that early cancer lesions invisible by the naked eye may be detected with the opto- acoustic tomography. Compact design of COAT allows, in principle, application of the opto-acoustic imaging in any organ of the human digestive system.

Cornea and ocular lens visualized with three-dimensional confocal microscopy

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1992-08-01

This paper demonstrates the advantages of three-dimensional reconstruction of the cornea and the ocular crystalline lens by confocal microscopy and volume rendering computer techniques. The advantages of noninvasive observation of ocular structures in living, unstained, unfixed tissue include the following: the tissue is in a natural living state without the artifacts of fixation, mechanical sectioning, and staining; the three-dimensional structure can be observed from any view point and quantitatively analyzed; the dynamics of morphological changes can be studied; and the use of confocal microscopic observation results in a reduction of the number of animals required for ocular morphometric studies. The main advantage is that the dynamic morphology of ocular structures can be investigated in living ocular tissue. A laser scanning confocal microscope was used in the reflected light mode to obtain the two- dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with 488 nm wavelength. The microscope objective was a Leitz 25X, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133, three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The under sampling resulted in a three-dimensional visualization rendering in which the corneal thickness (z-axis) is compressed. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their `beaded' cell borders, basal lamina, nerve plexus, nerve fibers, free nerve endings in the basal epithelial cells, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in-situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers.

Optical modeling of an ultrathin scanning fiber endoscope, a preliminary study of confocal versus non-confocal detection.

PubMed

Barhoum, Erek; Johnston, Richard; Seibel, Eric

2005-09-19

An optical model of an ultrathin scanning fiber endoscope was constructed using a non-sequential ray tracing program and used to study the relationship between fiber deflection and collection efficiency from tissue. The problem of low collection efficiency of confocal detection through the scanned single-mode optical fiber was compared to non-confocal cladding detection. Collection efficiency is 40x greater in the non-confocal versus the confocal geometry due to the majority of rays incident on the core being outside the numerical aperture. Across scan angles of 0 to 30o, collection efficiency decreases from 14.4% to 6.3% for the non-confocal design compared to 0.34% to 0.10% for the confocal design. Non-confocality provides higher and more uniform collection efficiencies at larger scan angles while sacrificing the confocal spatial filter.

Combined reflectance confocal microscopy-optical coherence tomography for delineation of basal cell carcinoma margins: an ex vivo study

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor; Peterson, Gary; Chang, Ernest W.; Maguluri, Gopi; Fox, William; Rajadhyaksha, Milind

2016-01-01

We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ˜1 μm, OCT imaging delineates BCC depth spreading (margins) with resolution of ˜7 μm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ˜600 μm, and agreement with histology was within about ±50 μm.

Narrow linewidth operation of a spectral beam combined diode laser bar.

PubMed

Zhu, Zhanda; Jiang, Menghua; Cheng, Siqi; Hui, Yongling; Lei, Hong; Li, Qiang

2016-04-20

Our experiment is expected to provide an approach for realizing ultranarrow linewidth for a spectral beam combined diode laser bar. The beams of a diode laser bar are combined in a fast axis after a beam transformation system. With the help of relay optics and a transform lens with a long focal length of 1.5 m, the whole wavelength of a spectral combined laser bar can be narrowed down to 0.48 nm from more than 10 nm. We have achieved 56.7 W cw from a 19-element single bar with an M² of 1.4  (in horizontal direction)×11.6  (in vertical direction). These parameters are good evidence that all the beams from the diode laser bar are combined together to increase the brightness.

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.

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

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.

Measure the spatial distribution of corneal elasticity by combining femtosecond laser induced breakdown spectroscopy and acoustic radiation force elasticity microscope

NASA Astrophysics Data System (ADS)

Sun, Hui; Li, Xin; Hu, Mingyong

2017-08-01

The unique spatial distribution of corneal elasticity is shown by the nonhomogeneous structure of the cornea. It is critical to understanding how biomechanics control corneal stability and refraction and one way to do this job is non-invasive measurement of this distribution. Femtosecond laser pulses have the ability to induce optical breakdown and produced cavitation in the anterior and posterior cornea. A confocal ultrasonic transducer applied 6.5 ms acoustic radiation forcechirp bursts to the bubble at 1.5 MHz while monitoring bubble position using pulse-echoes at 20 MHz. The laser induced breakdown spectroscopy (LIBS) were measured in the anterior and posterior cornea with the plasmas that induced by the same femtosecond laser to see whether the laser induced plasmas signals will show relationship to Young's modulus.

Precision of 655nm Confocal Laser Profilometry for 3D surface texture characterisation of natural human enamel undergoing dietary acid mediated erosive wear.

PubMed

Mullan, F; Mylonas, P; Parkinson, C; Bartlett, D; Austin, R S

2018-03-01

To assess the precision of optical profilometry for characterising the 3D surface roughness of natural and polished human enamel in order to reliably quantify acid mediated surface roughness changes in human enamel. Forty-two enamel samples were prepared from extracted human molars and either polished flat or left unmodified. To investigate precision, the variability of thirty repeated measurements of five areas of one polished and one natural enamel sample was assessed using 655nm Confocal Laser Profilometry. Remaining samples were subjected to forty-five minutes orange juice erosion and microstructural changes were analysed using Sa roughness change (μm) and qualitatively using surface/subsurface confocal microscopy. Enamel surface profilometry from the selected areas revealed maximal precision of 5nm for polished enamel and 23nm for natural enamel. After erosion, the polished enamel revealed a 48% increase in mean (SD) Sa roughness of 0.10 (0.07)μm (P<0.05), whereas in contrast the natural enamel revealed a 45% decrease in mean (SD) roughness of -0.32 (0.42)μm (P<0.05). These data were supported by qualitative confocal images of the surface/subsurface enamel. This study demonstrates a method for precise surface texture measurement of natural human enamel. Measurement precision was superior for polished flat enamel in contrast to natural enamel however, natural enamel responds very differently to polished enamel when exposed to erosion challenges. Therefore, thus future studies characterising enamel surface changes following erosion on natural enamel may provide more clinically relevant responses in comparison to polished enamel. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

ConfocalVR: Immersive Visualization Applied to Confocal Microscopy.

PubMed

Stefani, Caroline; Lacy-Hulbert, Adam; Skillman, Thomas

2018-06-24

ConfocalVR is a virtual reality (VR) application created to improve the ability of researchers to study the complexity of cell architecture. Confocal microscopes take pictures of fluorescently labeled proteins or molecules at different focal planes to create a stack of 2D images throughout the specimen. Current software applications reconstruct the 3D image and render it as a 2D projection onto a computer screen where users need to rotate the image to expose the full 3D structure. This process is mentally taxing, breaks down if you stop the rotation, and does not take advantage of the eye's full field of view. ConfocalVR exploits consumer-grade virtual reality (VR) systems to fully immerse the user in the 3D cellular image. In this virtual environment the user can: 1) adjust image viewing parameters without leaving the virtual space, 2) reach out and grab the image to quickly rotate and scale the image to focus on key features, and 3) interact with other users in a shared virtual space enabling real-time collaborative exploration and discussion. We found that immersive VR technology allows the user to rapidly understand cellular architecture and protein or molecule distribution. We note that it is impossible to understand the value of immersive visualization without experiencing it first hand, so we encourage readers to get access to a VR system, download this software, and evaluate it for yourself. The ConfocalVR software is available for download at http://www.confocalvr.com, and is free for nonprofits. Copyright © 2018. Published by Elsevier Ltd.

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.

In vitro confocal imaging of the rabbit cornea.

PubMed

Masters, B R; Paddock, S

1990-05-01

We were able to observe in vitro the fine structure of the rabbit cornea using a laser scanning confocal microscope, especially in the regions between Descemet's membrane and the epithelial basal lamina. We observed submicrometre filaments throughout the stroma with high concentrations adjacent to Descemet's membrane, and found extensive interconnecting processes between stromal keratocytes. There are numerous regions containing nerve plexuses in the stroma. We found a deeply convoluted basal lamina adjacent to the epithelium, and observed regions containing junctions between endothelial cells in fluorescent images of rabbit corneas stained with the actin-specific compound fluorescein phalloidin.

A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

NASA Astrophysics Data System (ADS)

Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

2017-12-01

There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

Laser-Ultrasonic Testing and its Applications to Nuclear Reactor Internals

NASA Astrophysics Data System (ADS)

Ochiai, M.; Miura, T.; Yamamoto, S.

2008-02-01

A new nondestructive testing technique for surface-breaking microcracks in nuclear reactor components based on laser-ultrasonics is developed. Surface acoustic wave generated by Q-switched Nd:YAG laser and detected by frequency-stabilized long pulse laser coupled with confocal Fabry-Perot interferometer is used to detect and size the cracks. A frequency-domain signal processing is developed to realize accurate sizing capability. The laser-ultrasonic testing allows the detection of surface-breaking microcrack having a depth of less than 0.1 mm, and the measurement of their depth with an accuracy of 0.2 mm when the depth exceeds 0.5 mm including stress corrosion cracking. The laser-ultrasonic testing system combined with laser peening system, which is another laser-based maintenance technology to improve surface stress, for inner surface of small diameter tube is developed. The generation laser in the laser-ultrasonic testing system can be identical to the laser source of the laser peening. As an example operation of the system, the system firstly works as the laser-ultrasonic testing mode and tests the inner surface of the tube. If no cracks are detected, the system then changes its work mode to the laser peening and improves surface stress to prevent crack initiation. The first nuclear industrial application of the laser-ultrasonic testing system combined with the laser peening was completed in Japanese nuclear power plant in December 2004.

Synergistic skin heat shock protein expression in response to combined laser treatment with a diode laser and ablative fractional lasers.

PubMed

Paasch, Uwe; Sonja, Grunewald; Haedersdal, Merete

2014-06-01

Diode laser-based skin heating has been shown to minimise scars by interfering with wound healing responses through the induction of heat shock proteins (HSP). HSP are also induced after ablative fractional laser (AFXL) wound healing. AFXL itself is highly recommended for scar treatment. Therefore, the sequential combination of both modalities may produce superior outcomes. The aim of this study was to examine the pretreatment effects of a diode laser before AFXL on wound healing responses in terms of HSP up-regulation in an in vitro model. Immediate responses and responses on days 1, 3 or 6 post-procedure were studied in an in vitro porcine skin model (n = 240). Untreated samples served as control. Immunohistochemical investigation (Hsp70) was performed in all untreated controls, diode laser-, AFXL-, and in diode laser + AFXL-treated samples. Hsp70 was shown to be up-regulated by all interventions between days 1 and 6 after interventions. The largest effect was caused by the combination of a diode laser and an AFXL procedure. Diode laser exposure induces a skin HSP response that can be further enhanced by sequential AFXL treatment. Clinical studies are necessary to investigate the dose response of HSP on scar formation and refine suitable laser exposure settings.

In-situ Crystallization of Highly Volatile Commercial Mold Flux Using an Isolated Observation System in the Confocal Laser Scanning Microscope

NASA Astrophysics Data System (ADS)

Park, Jun-Yong; Ryu, Jae Wook; Sohn, Il

2014-08-01

The in situ crystallization behavior of highly volatile commercial mold fluxes for medium carbon steels was investigated using the confocal laser scanning microscope (CLSM) equipped with an optimized isolated observation system. The highly volatile compounds of the mold flux were suppressed during heating allowing direct observation in the CLSM. Cooling rates of 25, 50, 100, 400, and 800 K/min were incorporated and continuous cooling transformation (CCT) diagrams of 4 different commercial mold fluxes for medium carbon steels were developed. Identification of the crystalline phase was conducted with XRD and SEM-EDS analysis. A cuspidine crystalline was observed in all samples at various cooling rates. With higher basicity, CaF2, and NaF, the crystallization of the fluxes was enhanced according to the CCT diagram. As the slag structure becomes depolymerized, the diffusion rate of the cathodic ions seems to increase.

The effectiveness and safety of combining laser-assisted liposuction and abdominoplasty.

PubMed

Aboelatta, Yasser Abdallah; Abdelaal, Mohammed Mahmoud; Bersy, Nada Abdelsatar

2014-02-01

Lipoabdominoplasty is nearly a daily aesthetic procedure. Despite the emergence of laser-assisted liposuction, to date, it has not been clearly evaluated combined with abdominoplasty. This prospective study aimed to evaluate the effectiveness and safety of laser-assisted liposuction relative to traditional liposuction combined with high-lateral-tension abdominoplasty. This study investigated 36 consecutive female patients who underwent high-lateral-tension abdominoplasty combined with liposuction of the upper central abdomen and both flanks. The patients were divided into three equal groups based on the technique used for liposuction. Group 1 underwent conventional liposuction with abdominoplasty. Group 2 underwent a mixture of conventional and laser-assisted liposuction with abdominoplasty. Group 3 underwent laser-assisted liposuction with abdominoplasty. The patients in groups 2 and 3 had a better aesthetic outcome than those in group 1 with regard to abdominal contour and skin tightness. No major complications were observed in groups 1 and 2. The patients in group 3 had a higher incidence of complications (3 seromas, 3 central necroses and dehiscence), and one patient underwent secondary sutures. Laser-assisted liposuction combined with abdominoplasty in the lateral abdomen seems to be a safe technique with good aesthetic outcomes. Although the combined use of laser-assisted liposuction in the lateral and central abdomen can achieve relatively better aesthetic results, it is associated with significant complications, and its use cannot be supported. Proper laser parameters in the central abdominal area still need further study. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

[Confocal laser scanning electron microscopy for assessment of vaginal Lactobacillus crispatus biofilm].

PubMed

Wu, Li-jie; Wang, Ben; Liao, Qin-ping; Zhang, Rui

2015-12-18

To investigate the female vaginal Lactobacillus crispatus biofilm by using confocal laser scanning microscopy (CLSM),thus revealing the formation of biofilm. The cover slide biofilm culture approach in vitro was employed for induction of the vaginal Lactobacillus crispatus biofilm formation. Following the culture for 2, 4, 8, 12, 16, 20, 24, 48, 72, 96 and 120 hours, the cover slide was removed for subsequent staining with the fluoresce in isothiocyanate-conjugated concanavalin A(FITC-ConA) and propidium (PI).This was followed by determination of the formation and characteristics of the vaginal Lactobacillus crispatus biofilm by using CLSM. The CLSM images of biofilm formation at different time points were captured, suggesting that the vaginal Lactobacillus crispatus adhesion occurred at h 4, which was in reversible attachment, then more and more Lactobacillus crispatus aggregated at h 8 to h 20, which was in irreversible attachment.Lactobacillus crispatus clustered at h 20, with early development of biofilm architecture.Then the biofilm with extracellular matrix around the bacteria was set up at h 24,with gradual matureation at h 24 to h 48.The biofilm dispersed at h 72. The biofilm density of cultivating for 20 hours was 42.7 × 10⁻³ ± 6.8 × 10⁻³ ,and for 24 hours increased to 102.5 × 10⁻³ ± 23.1 × 10⁻³, suggesting a significant difference, P<0.05. This meant that mature biofilm was formed at h 24. The vaginal Lactobacillus crispatus is able to form typical biofilm with distinct developmental phases and architecture characteristics.Mature biofilm is formed at h 24 to h 48, then the biofilm begins to disperse.

Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas.

PubMed

Sanai, Nader; Snyder, Laura A; Honea, Norissa J; Coons, Stephen W; Eschbacher, Jennifer M; Smith, Kris A; Spetzler, Robert F

2011-10-01

Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)-induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Intraoperative confocal microscopy can visualize cellular 5-ALA-induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

Confocal Raman Microscopy: new perspective on the weathering of anhydrous cement

NASA Astrophysics Data System (ADS)

Torres-Carrasco, M.; del Campo, A.; de la Rubia, MA; Reyes, E.; Moragues, A.; Fernández, JF

2017-10-01

Raman spectroscopy when is combined with Confocal microscopy is a non-destructive technique that allow us to obtain information in cementitious materials. In this study, we present non-destructive image and structural analysis of anhydrous cement with carbonation evidences by Confocal Raman Microscopy (CRM). The results obtained by CRM show a direct relationship between the presence of the weathering processes of an anhydrous cement with the presence of sulphates and surprisingly, with the existence of amorphous carbon in the medium.

Combination treatment with excimer laser and narrowband UVB light in vitiligo patients.

PubMed

Shin, Sungsik; Hann, Seung-Kyung; Oh, Sang Ho

2016-01-01

For the treatment of vitiligo, narrowband UVB (NBUVB) light is considered the most effective for nonsegmental vitiligo, while excimer laser treatment is commonly used for localized vitiligo. However, treatment areas may potentially be missed with excimer laser treatment. We aimed to evaluate the effect of combinational treatment with NBUVB light and excimer laser on vitiligo. All patients were first treated with NBUVB; excimer laser was then applied in conjunction with NBUVB phototherapy due to a slow response or no further improvement with continuous NBUVB treatment alone. To minimize adverse effects, a fixed dose of NBUVB was administered, and the dose of excimer laser was increased based on patient response. Among 80 patients, 54 patients showed responses after combination with excimer laser; however, 26 patients (32.5%) showed no remarkable change after combination therapy. Of the 26 patients who showed no further response, 12 patients (46.1%) presented with vitiligo on the acral areas, which are known to the least responsive sites. Our study suggests that combined treatment of NBUVB and excimer laser in vitiligo may enhance the treatment response without remarkable side effects, therefore might also increase the compliance of the patients to the treatment. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Confocal micro-Raman spectroscopy of single biological cells using optical trapping and shifted excitation difference techniques

NASA Astrophysics Data System (ADS)

Xie, Changan; Li, Yong-qing

2003-03-01

We report on the study of single biological cells with a confocal micro-Raman spectroscopy system that uses optical trapping and shifted excitation Raman difference technique. A tunable diode laser was used to capture a living cell in solution, confine it in the confocal excitation volume, and then excite the Raman scattering. The optical trapping allows us to lift the cell well off the cover plate so that the fluorescence interference from the plate can be effectively reduced. In order to further remove the interference of the fluorescence and stray light from the trapped cell, we employed a shifted excitation Raman difference technique with slightly tuned laser frequencies. With this system, high-quality Raman spectra were obtained from single optically trapped biological cells including E. coli bacteria, yeast cells, and red blood cells. A significant difference between control and heat-treated E. coli B cells was observed due to the denaturation of biomolecules.

High efficiency pump combiner fabricated by CO2 laser splicing system

NASA Astrophysics Data System (ADS)

Zhu, Gongwen

2018-02-01

High power combiners are of great interest for high power fiber lasers and fiber amplifiers. With the advent of CO2 laser splicing system, power combiners are made possible with low manufacturing cost, low loss, high reliability and high performance. Traditionally fiber optical components are fabricated with flame torch, electrode arc discharge or filament heater. However, these methods can easily leave contamination on the fiber, resulting inconsistent performance or even catching fire in high power operations. The electrodes or filaments also degrade rapidly during the combiner manufacturing process. The rapid degradation will lead to extensive maintenance, making it unpractical or uneconomic for volume production. By contrast, CO2 laser is the cleanest heating source which provides reliable and repeatable process for fabricating fiber optic components including high power combiners. In this paper we present an all fiber end pumped 7x1 pump combiner fabricated by CO2 laser splicing system. The input pump fibers are 105/125 (core/clad diameters in μm) fibers with a core NA of 0.22. The output fiber is a 300/320 fiber with a core NA of 0.22. The average efficiency is 99.4% with all 7 ports more than 99%. The process is contamination-free and highly repeatable. To our best knowledge, this is the first report in the literature on power combiners fabricated by CO2 laser splicing system. It also has the highest reported efficiency of its kind.

Generating a high brightness multi-kilowatt laser by dense spectral combination of VBG stabilized single emitter laser diodes

NASA Astrophysics Data System (ADS)

Fritsche, H.; Koch, Ralf; Krusche, B.; Ferrario, F.; Grohe, Andreas; Pflueger, S.; Gries, W.

2014-05-01

Generating high power laser radiation with diode lasers is commonly realized by geometrical stacking of diode bars, which results in high output power but poor beam parameter product (BPP). The accessible brightness in this approach is limited by the fill factor, both in slow and fast axis. By using a geometry that accesses the BPP of the individual diodes, generating a multi kilowatt diode laser with a BPP comparable to fiber lasers is possible. We will demonstrate such a modular approach for generating multi kilowatt lasers by combining single emitter diode lasers. Single emitter diodes have advantages over bars, mainly a simplified cooling, better reliability and a higher brightness per emitter. Additionally, because single emitters can be arranged in many different geometries, they allow building laser modules where the brightness of the single emitters is preserved. In order to maintain the high brightness of the single emitter we developed a modular laser design which uses single emitters in a staircase arrangement, then coupling two of those bases with polarization combination which is our basic module. Those modules generate up to 160 W with a BPP better than 7.5 mm*mrad. For further power scaling wavelength stabilization is crucial. The wavelength is stabilized with only one Volume Bragg Grating (VBG) in front of a base providing the very same feedback to all of the laser diodes. This results in a bandwidth of < 0.5 nm and a wavelength stability of better than 250 MHz over one hour. Dense spectral combination with dichroic mirrors and narrow channel spacing allows us to combine multiple wavelength channels, resulting in a 2 kW laser module with a BPP better than 7.5 mm*mrad, which can easily coupled into a 100 μm fiber and 0.15 NA.

Chemical imaging of drug delivery systems with structured surfaces-a combined analytical approach of confocal raman microscopy and optical profilometry.

PubMed

Kann, Birthe; Windbergs, Maike

2013-04-01

Confocal Raman microscopy is an analytical technique with a steadily increasing impact in the field of pharmaceutics as the instrumental setup allows for nondestructive visualization of component distribution within drug delivery systems. Here, the attention is mainly focused on classic solid carrier systems like tablets, pellets, or extrudates. Due to the opacity of these systems, Raman analysis is restricted either to exterior surfaces or cross sections. As Raman spectra are only recorded from one focal plane at a time, the sample is usually altered to create a smooth and even surface. However, this manipulation can lead to misinterpretation of the analytical results. Here, we present a trendsetting approach to overcome these analytical pitfalls with a combination of confocal Raman microscopy and optical profilometry. By acquiring a topography profile of the sample area of interest prior to Raman spectroscopy, the profile height information allowed to level the focal plane to the sample surface for each spectrum acquisition. We first demonstrated the basic principle of this complementary approach in a case study using a tilted silica wafer. In a second step, we successfully adapted the two techniques to investigate an extrudate and a lyophilisate as two exemplary solid drug carrier systems. Component distribution analysis with the novel analytical approach was neither hampered by the curvature of the cylindrical extrudate nor the highly structured surface of the lyophilisate. Therefore, the combined analytical approach bears a great potential to be implemented in diversified fields of pharmaceutical sciences.

Fabrication of multi-scale periodic surface structures on Ti-6Al-4V by direct laser writing and direct laser interference patterning for modified wettability applications

NASA Astrophysics Data System (ADS)

Huerta-Murillo, D.; Aguilar-Morales, A. I.; Alamri, S.; Cardoso, J. T.; Jagdheesh, R.; Lasagni, A. F.; Ocaña, J. L.

2017-11-01

In this work, hierarchical surface patterns fabricated on Ti-6Al-4V alloy combining two laser micro-machining techniques are presented. The used technologies are based on nanosecond Direct Laser Writing and picosecond Direct Laser Interference Patterning. Squared shape micro-cells with different hatch distances were produced by Direct Laser Writing with depths values in the micro-scale, forming a well-defined closed packet. Subsequently, cross-like periodic patterns were fabricated by means of Direct Laser Interference Patterning using a two-beam configuration, generating a dual-scale periodic surface structure in both micro- and nano-scale due to the formation of Laser-Induced Periodic Surface Structure after the picosecond process. As a result a triple hierarchical periodic surface structure was generated. The surface morphology of the irradiated area was characterized with scanning electron microscopy and confocal microscopy. Additionally, static contact angle measurements were made to analyze the wettability behavior of the structures, showing a hydrophobic behavior for the hierarchical structures.

Fluorescence imaging of reactive oxygen species by confocal laser scanning microscopy for track analysis of synchrotron X-ray photoelectric nanoradiator dose: X-ray pump-optical probe.

PubMed

Jeon, Jae Kun; Han, Sung Mi; Kim, Jong Ki

2016-09-01

penetration by nanoradiators. In conclusion, the combined use of a synchrotron X-ray microbeam-irradiated three-dimensional ROS gel and confocal laser scanning fluorescence microscopy provides a simple dosimetry method for track analysis of X-ray photoelectric nanoradiator radiation, suggesting extensive cellular damage with dose-enhancement beyond a single cell containing IONs.

Cascaded injection resonator for coherent beam combining of laser arrays

DOEpatents

Kireev, Vassili [Sunnyvale, CA; Liu, Yun; Protopopescu, Vladimir [Knoxville, TN; Braiman, Yehuda [Oak Ridge, TN

2008-10-21

The invention provides a cascaded injection resonator for coherent beam combining of laser arrays. The resonator comprises a plurality of laser emitters arranged along at least one plane and a beam sampler for reflecting at least a portion of each laser beam that impinges on the beam sampler, the portion of each laser beam from one of the laser emitters being reflected back to another one of the laser emitters to cause a beam to be generated from the other one of the laser emitters to the beam reflector. The beam sampler also transmits a portion of each laser beam to produce a laser output beam such that a plurality of laser output beams of the same frequency are produced. An injection laser beam is directed to a first laser emitter to begin a process of generating and reflecting a laser beam from one laser emitter to another laser emitter in the plurality. A method of practicing the invention is also disclosed.

High-power beam combining: a step to a future laser weapon system

NASA Astrophysics Data System (ADS)

Protz, Rudolf; Zoz, Jürgen; Geidek, Franz; Dietrich, Stephan; Fall, Michael

2012-11-01

Due to the enormous progress in the field of high-power fiber lasers during the last years commercial industrial fiber lasers are now available, which deliver a near-diffraction limited beam with power levels up to10kW. For the realization of a future laser weapon system, which can be used for Counter-RAM or similar air defence applications, a laser source with a beam power at the level of 100kW or more is required. At MBDA Germany the concept for a high-energy laser weapon system is investigated, which is based on such existing industrial laser sources as mentioned before. A number of individual high-power fiber laser beams are combined together, using one common beam director telescope. By this "geometric" beam coupling scheme, sufficient laser beam power for an operational laser weapon system can be achieved. The individual beams from the different lasers are steered by servo-loops, using fast tip-tilt mirrors. This principle enables the concentration of the total laser beam power at the common focal point on a distant target, also allowing fine tracking of target movements and first order compensation of turbulence effects on laser beam propagation. The proposed beam combination concept was demonstrated using several experimental set-ups. Different experiments were performed, to investigate laser beam target interaction and target fine tracking also at large distances. Content and results of these investigations are reported. An example for the lay-out of an Air Defence High Energy Laser Weapon (ADHELW ) is given. It can be concluded, that geometric high-power beam combining is an important step for the realization of a laser weapon system in the near future.

Theoretical investigation of confocal microscopy using an elliptically polarized cylindrical vector laser beam: Visualization of quantum emitters near interfaces

NASA Astrophysics Data System (ADS)

Boichenko, Stepan

2018-04-01

We theoretically study laser-scanning confocal fluorescence microscopy using elliptically polarized cylindrical vector excitation light as a tool for visualization of arbitrarily oriented single quantum dipole emitters located (1) near planar surfaces enhancing fluorescence, (2) in a thin supported polymer film, (3) in a freestanding polymer film, and (4) in a dielectric planar microcavity. It is shown analytically that by using a tightly focused azimuthally polarized beam, it is possible to exclude completely the orientational dependence of the image intensity maximum of a quantum emitter that absorbs light as a pair of incoherent independent linear dipoles. For linear dipole quantum emitters, the orientational independence degree higher than 0.9 can normally be achieved (this quantity equal to 1 corresponds to completely excluded orientational dependence) if the collection efficiency of the microscope objective and the emitter's total quantum yield are not strongly orientationally dependent. Thus, the visualization of arbitrarily oriented single quantum emitters by means of the studied technique can be performed quite efficiently.

Novel approach to real-time flash photolysis and confocal [Ca2+] imaging

PubMed Central

Sobie, Eric A.; Kao, Joseph P.Y.; Lederer, W. J.

2008-01-01

Flash photolysis of “caged” compounds using ultraviolet light is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. Studies that employ this technique have used diverse strategies for controlling the spatial and temporal application of light to the specimen. Here we describe a new system for flash photolysis that delivers light from a pulsed, adjustable intensity laser through an optical fiber coupled into the epifluorescence port of a commercial confocal microscope. Photolysis is achieved with extremely brief (5 ns) pulses of ultraviolet light (355 nm) that can be synchronized with respect to confocal laser scanning. The system described also localizes the UV intensity spatially so that uncaging only occurs in defined sub-cellular regions; moreover, since the microscope optics are used in localization, the photolysis volume can be easily adjusted. Experiments performed on rat ventricular myocytes loaded with the Ca2+ indicator fluo-3 and the Ca2+ cage NP-EGTA demonstrate the system's capabilities. Localized intracellular increases in [Ca2+] can trigger sarcoplasmic reticular Ca2+ release events such as Ca2+ sparks and, under certain conditions, regenerative Ca2+ waves. This relatively simple and inexpensive system is therefore a useful tool for examining local signaling in heart and other tissues. PMID:17323075

High resolution 3D confocal microscope imaging of volcanic ash particles.

PubMed

Wertheim, David; Gillmore, Gavin; Gill, Ian; Petford, Nick

2017-07-15

We present initial results from a novel high resolution confocal microscopy study of the 3D surface structure of volcanic ash particles from two recent explosive basaltic eruptions, Eyjafjallajökull (2010) and Grimsvötn (2011), in Iceland. The majority of particles imaged are less than 100μm in size and include PM 10 s, known to be harmful to humans if inhaled. Previous studies have mainly used 2D microscopy to examine volcanic particles. The aim of this study was to test the potential of 3D laser scanning confocal microscopy as a reliable analysis tool for these materials and if so to what degree high resolution surface and volume data could be obtained that would further aid in their classification. First results obtained using an Olympus LEXT scanning confocal microscope with a ×50 and ×100 objective lens are highly encouraging. They reveal a range of discrete particle types characterised by sharp or concave edges consistent with explosive formation and sudden rupture of magma. Initial surface area/volume ratios are given that may prove useful in subsequent modelling of damage to aircraft engines and human tissue where inhalation has occurred. Copyright © 2017 Elsevier B.V. All rights reserved.

Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

PubMed

Okuno, Masanari; Hamaguchi, Hiro-o

2010-12-15

We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

Facelift combined with simultaneous fractional laser resurfacing: Outcomes and complications.

PubMed

Wright, Eric J; Struck, Steve K

2015-10-01

The combination of simultaneous surgical rhytidectomy with ablative resurfacing has been a controversial procedure due to the concern of postoperative wound healing. Traditional ablative resurfacing lasers are believed to have higher rates of complications, leading to delayed healing and skin flap loss when combined with face rhytidectomy surgeries. With the development of fractionated ablative laser therapy, there has been increased interest in combining these two procedures. The objective of this study is to evaluate the clinical outcomes of patients undergoing simultaneous full-face rhytidectomy in combination with fractionated ablative skin resurfacing. A retrospective chart analysis was performed for all patients who had a combined procedure of facelift and ablative fractional laser resurfacing from 2008 to 2013 by the senior author (SKS). Postoperative recovery and complications were recorded. The surgical technique used for performing the facelift was an extended supraplatysmal dissection with SMAS plication. Fraxel Re:Pair 10,600-nm fractional carbon dioxide laser was used to perform an ablative resurfacing including the elevated skin flaps. A total of 86 patients were included. Average age was 60.01 years (range of 45-78 years). Longest follow up was five years. The average size of the elevated skin flaps was 100 cm(2). Average skin type was a Fitzpatrick type 2. All patients had complete re-epithelialization by one week after their procedure. Four patients (4.6%) experienced acne outbreaks. Four patients (4.6%) had facial erythema that persisted greater than two weeks. Of these four patients, all resolved by five weeks postoperatively. There was no delayed wound healing or skin flap loss observed. Our results indicate that simultaneous rhytidectomy with fractionated ablative laser resurfacing does not cause an increase in wound healing or skin loss. Due to improved patient outcomes with combining these procedures, we believe that this can be increasingly

Laser stimulation can activate autophagy in HeLa cells

NASA Astrophysics Data System (ADS)

Wang, Yisen; Lan, Bei; He, Hao; Hu, Minglie; Cao, Youjia; Wang, Chingyue

2014-10-01

For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flash of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca2+ dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.

Laser stimulation can activate autophagy in HeLa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yisen; Hu, Minglie; Wang, Chingyue

For decades, lasers have been a daily tool in most biological research for fluorescent excitation by confocal or multiphoton microscopy. More than 20 years ago, cell photodamage caused by intense laser stimulation was noticed by generating reactive oxygen species, which was then thought as the main damage effect by photons. In this study, we show that laser stimulation can induce autophagy, an important cell lysosomal pathway responding to immune stimulation and starvation, without any biochemical treatment. Two different types of laser stimulations are found to be capable of activating autophagy: continuous scanning by continuous-wave visible lasers and a short-time flashmore » of femtosecond laser irradiation. The autophagy generation is independent from wavelength, power, and scanning duration of the visible lasers. In contrast, the power of femtosecond laser is very critical to autophagy because the multiphoton excited Ca{sup 2+} dominates autophagy signaling. In general, we show here the different mechanisms of autophagy generation by such laser stimulation, which correspond to confocal microscopy and cell surgery, respectively. Those results can help further understanding of photodamage and autophagy signaling.« less

Confocal Laser Endomicroscopy for the Diagnosis of Urothelial Carcinoma in the Bladder and the Upper Urinary Tract: Protocols for Two Prospective Explorative Studies.

PubMed

Liem, Esmee Iml; Freund, Jan Erik; Baard, Joyce; de Bruin, D Martijn; Laguna Pes, M Pilar; Savci-Heijink, C Dilara; van Leeuwen, Ton G; de Reijke, Theo M; de la Rosette, Jean Jmch

2018-02-07

Visual confirmation of a suspicious lesion in the urinary tract is a major corner stone in diagnosing urothelial carcinoma. However, during cystoscopy (for bladder tumors) and ureterorenoscopy (for tumors of the upper urinary tract) no real-time histopathologic information can be obtained. Confocal laser endomicroscopy (CLE) is an optical imaging technique that allows for in vivo high-resolution imaging and may allow real-time tumor grading of urothelial lesions. The primary objective of both studies is to develop descriptive criteria for in vivo CLE images of urothelial carcinoma (low-grade, high-grade, carcinoma in situ) and normal urothelium by comparing CLE images with corresponding histopathology. In these two prospective clinical trials, CLE imaging will be performed of suspicious lesions and normal tissue in the urinary tract during surgery, prior to resection or biopsy. In the bladder study, CLE will be performed in 60 patients using the Cystoflex UHD-R probe. In the upper urinary tract study, CLE will be performed in 25 patients during ureterorenoscopy, who will undergo radical treatment (nephroureterectomy or segmental ureter resection) thereafter. All CLE images will be analyzed frame by frame by three independent, blinded observers. Histopathology and CLE-based diagnosis of the lesions will be evaluated. Both studies comply with the IDEAL stage 2b recommendations. Presently, recruitment of patients is ongoing in both studies. Results and outcomes are expected in 2018. For development of CLE-based diagnosis of urothelial carcinoma in the bladder and the upper urinary tract, a structured conduct of research is required. This study will provide more insight in tissue-specific CLE criteria for real-time tumor grading of urothelial carcinoma. Confocal Laser Endomicroscopy: ClinicalTrials.gov NCT03013894; https://clinicaltrials.gov /ct2/show/NCT03013894?term=NCT03013894&rank=1 (Archived by WebCite at http://www.webcitation.org/6wiPZ378I); and Dutch Central

Combined pulsed dye laser and fiberoptic Nd-YAG laser for the treatment of hypertrophic port wine stain.

PubMed

Radmanesh, Mohammed; Radmanesh, Ramin

2017-10-01

The hypertrophic Port Wine Stain (PWS) is only partially and superficially treated with the Pulsed dye laser (PDL) because of its limited depth of penetration. We used combined PDL and fiberoptic 1444-nm Nd-YAG laser to treat a case with hypertrophic PWS. After tumescent anesthesia, few holes were made by a 16-gauge needle on different sides of the lesion. The fiberoptic tip of 1444-nm Nd-YAG laser was inserted within the holes and was pushed forward while triggering. In a fan pattern and by a back and forth movement, the subcutaneous and deep dermal areas were coagulated. The skin and outer mucosal surfaces were then treated by PDL. The fiberoptic system used was Accusculpt 1444-nm Nd-YAG laser (Lutronic lasers, South Korea), and the PDL used was 585 nm Nlite system (Chromogenex UK). The parameters used for PDL were fluence = 9 Joules/cm 2 and the spot size was 5 mm. The parameters used for fiberoptic 1444-nm Nd-YAG laser were: Pulse rate = 30 Hz, pulse energy = 300 mJ, power = 6 W, and the total energy = 4000 J for the whole face and mucosa. Little sign of regression and moderate purpura were detected immediately after combined fiberoptic Nd-YAG and PDL therapy. The lesion gradually regressed within 4 months with satisfactory color and volume change. Combined fiberoptic Nd-YAG laser and PDL can be used for the treatment of deeper and superficial layers of hypertrophic PWS.

Crystallization Behavior of Perovskite in the Synthesized High-Titanium-Bearing Blast Furnace Slag Using Confocal Scanning Laser Microscope

NASA Astrophysics Data System (ADS)

Hu, Meilong; Liu, Lu; Lv, Xuewei; Bai, Chenguang; Zhang, Shengfu

2014-01-01

The isothermal phase composition of high-titanium-bearing slag (23 mass pct TiO2) under an argon atmosphere during cooling process from 1723 K (1450 °C) was calculated by FactSage.6.3 (CRCT-ThermFact Inc., Montréal, Canada). Three main phases, which were perovskite, titania spinel, and clinopyroxene, could form during the cooling process and they precipitated at 1713 K, 1603 K, and 1498 K (1440 °C, 1330 °C, and 1225 °C), respectively. The nonisothermal crystallization process of perovskite in synthesized high-titanium-bearing slag was studied in situ by a confocal scanning laser microscope (CSLM) with cooling rate of 30 K/min. The results showed that the primary phase was perovskite that precipitated at 1703 K (1430 °C). The whole precipitation and growth process of perovskite was obtained, whereas other phases formed as glass under the current experimental conditions. Perovskite grew along a specific growth track and finally appeared with snowflake morphology. The growing kinetics of perovskite formation from molten slag were also mentioned.

A laser scanning confocal imaging-surface plasmon resonance system application in real time detection of antibody-antigen interaction

NASA Astrophysics Data System (ADS)

Zhang, H. Y.; Yang, L. Q.; Liu, W. M.

2011-12-01

The laser scanning confocal microscope (LSCM) offers several advantages over conventional optical microscopy, but most LSCM work is qualitative analysis and it is very hard to achieve quantitative detection directly with the changing of the fluorescent intensity. A new real time sensor system for the antibody-antigen interaction detection was built integrating with a LSCM and a wavelength-dependent surface plasmon resonance (SPR) sensor. The system was applied to detect the bonding process of human IgG and fluorescent-labeled affinity purified antibody in real time. The fluorescence images changing is well with that of SPR wavelengths in real time, and the trend of the resonance wavelength shift with the concentrations of antibody is similar to that of the fluorescent intensity changing. The results show that SPR makes up the short of quantificational analysis with LSCM with the high spatial resolution. The sensor system shows the merits of the of the LSCM and SPR synergetic application, which are of great importance for practical application in biosensor and life science for interesting local interaction.

Experimental Study of 5-fluorouracil Encapsulated Ethosomes Combined with CO2 Fractional Laser to Treat Hypertrophic Scar.

PubMed

Zhang, Zhen; Chen, Jun; Huang, Jun; Wo, Yan; Zhang, Yixin; Chen, Xiangdong

2018-01-18

This study is designed to explore permeability of ethosomes encapsulated with 5-florouracil (5-FU) mediated by CO 2 fractional laser on hypertrophic scar tissues. Moreover, therapeutic and duration effect of CO 2 fractional laser combined with 5-FU encapsulated ethosomes in rabbit ear hypertrophic scar model will be evaluated. The permeated amount of 5-FU and retention contents of 5-FU were both determined by high-performance liquid chromatography (HPLC). Fluorescence intensities of ethosomes encapsulated with 5-FU (5E) labeled with Rodanmin 6GO (Rho) were measured by confocal laser scanning microscopy (CLSM). The permeability promotion of 5E labeled with Rho in rabbit ear hypertrophic scar mediated by CO 2 fractional laser was evaluated at 0 h, 6 h, 12 h, 24 h, 3 days and 7 days after the irradiation. The opening rates of the micro-channels were calculated according to CLSM. The therapeutic effect of 5EL was evaluated on rabbit ear hypertrophic scar in vivo. Relative thickness of rabbit ear hypertrophic scar before and after the treatment was measured by caliper method. Scar elevation index (SEI) of rabbit ear hypertrophic scar was measured using H&E staining. The data showed that the penetration amount of 5EL group was higher than 5E group (4.15 ± 2.22 vs. 0.73 ± 0.33; p < 0.05) after 1-h treatment. Additionally, the penetration amount of 5EL was higher than that of the 5E group (107.61 ± 13.27 vs. 20.73 ± 3.77; p < 0.05) after 24-h treatment. The retention contents of the 5EL group also showed higher level than 5E group (24.42 ± 4.37 vs.12.25 ± 1.64; p < 0.05). The fluorescence intensity of Rho in hypertrophic scar tissues of the 5EL group was higher than that of the 5E group at different time points (1, 6, and 24 h). The opening rates of the micro-channels were decreased gradually within 24 h, and micro-channels were closed completely 3 days after the irradiation by CO 2 fractional laser. The relative thickness

Adaptive thresholding image series from fluorescence confocal scanning laser microscope using orientation intensity profiles

NASA Astrophysics Data System (ADS)

Feng, Judy J.; Ip, Horace H.; Cheng, Shuk H.

2004-05-01

Many grey-level thresholding methods based on histogram or other statistic information about the interest image such as maximum entropy and so on have been proposed in the past. However, most methods based on statistic analysis of the images concerned little about the characteristics of morphology of interest objects, which sometimes could provide very important indication which can help to find the optimum threshold, especially for those organisms which have special texture morphologies such as vasculature, neuro-network etc. in medical imaging. In this paper, we propose a novel method for thresholding the fluorescent vasculature image series recorded from Confocal Scanning Laser Microscope. After extracting the basic orientation of the slice of vessels inside a sub-region partitioned from the images, we analysis the intensity profiles perpendicular to the vessel orientation to get the reasonable initial threshold for each region. Then the threshold values of those regions near the interest one both in x-y and optical directions have been referenced to get the final result of thresholds of the region, which makes the whole stack of images look more continuous. The resulting images are characterized by suppressing both noise and non-interest tissues conglutinated to vessels, while improving the vessel connectivities and edge definitions. The value of the method for idealized thresholding the fluorescence images of biological objects is demonstrated by a comparison of the results of 3D vascular reconstruction.

Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection.

PubMed

Weaver, Mitchell T; Lynch, Kyle B; Zhu, Zaifang; Chen, Huang; Lu, Joann J; Pu, Qiaosheng; Liu, Shaorong

2017-04-01

Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary - hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments. Copyright © 2016 Elsevier B.V. All rights reserved.

Conjugation of both on-axis and off-axis light in Nipkow disk confocal microscope to increase availability of incoherent light source.

PubMed

Saito, Kenta; Arai, Yoshiyuki; Zhang, Jize; Kobayashi, Kentaro; Tani, Tomomi; Nagai, Takeharu

2011-01-01

Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.

3D Image Analysis of Geomaterials using Confocal Microscopy

NASA Astrophysics Data System (ADS)

Mulukutla, G.; Proussevitch, A.; Sahagian, D.

2009-05-01

Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the

Experimental demonstration of passive coherent combining of fiber lasers by phase contrast filtering.

PubMed

Jeux, François; Desfarges-Berthelemot, Agnès; Kermène, Vincent; Barthelemy, Alain

2012-12-17

We report experiments on a new laser architecture involving phase contrast filtering to coherently combine an array of fiber lasers. We demonstrate that the new technique yields a more stable phase-locking than standard methods using only amplitude filtering. A spectral analysis of the output beams shows that the new scheme generates more resonant frequencies common to the coupled lasers. This property can enhance the combining efficiency when the number of lasers to be coupled is large.

Use of stereo vision and 24-bit false-color imagery to enhance visualization of multimodal confocal images

NASA Astrophysics Data System (ADS)

Beltrame, Francesco; Diaspro, Alberto; Fato, Marco; Martin, I.; Ramoino, Paola; Sobel, Irwin E.

1995-03-01

Confocal microscopy systems can be linked to 3D data oriented devices for the interactive navigation of the operator through a 3D object space. Sometimes, such environments are named `virtual reality' or `augmented reality' systems. We consider optical confocal laser scanning microscopy images, in fluorescence with various excitations and emissions, and versus time The aim of our study has been the quantitative spatial analysis of confocal data using the false-color composition technique. Starting from three 2D confocal fluorescent images at the same slice location in a given biological specimen, a new single image representation of all three parameters has been generated by the false-color technique on a HP 9000/735 workstation, connected to the confocal microscope. The color composite result of the mapping of the three parameters is displayed using a resolution of 24 bits per pixel. The operator may independently vary the mix of each of the three components in the false-color composite via three (R, G, B) mixing sliders. Furthermore, by using the pixel data in the three fluorescent component images, a 3D space containing the density distribution of these three parameters has been constructed. The histogram has been displayed in stereo: it can be used for clustering purposes from the operator, through an original thresholding algorithm.

Planarization of Isolated Defects on ICF Target Capsule Surfaces by Pulsed Laser Ablation

DOE PAGES

Alfonso, Noel; Carlson, Lane C.; Bunn, Thomas L.

2016-08-09

Demanding surface quality requirements for inertial confinement fusion (ICF) capsules motivated the development of a pulsed laser ablation method to reduce or eliminate undesirable surface defects. The pulsed laser ablation technique takes advantage of a full surface (4π) capsule manipulation system working in combination with an optical profiling (confocal) microscope. Based on the defect topography, the material removal rate, the laser pulse energy and its beam profile, a customized laser raster pattern is derived to remove the defect. The pattern is a table of coordinates and number of pulses that dictate how the defect will be vaporized until its heightmore » is level with the capsule surface. This paper explains how the raster patterns are optimized to minimize surface roughness and how surface roughness after laser ablation is simulated. The simulated surfaces are compared with actual ablated surfaces. Large defects are reduced to a size regime where a tumble finishing process produces very high quality surfaces devoid of high mode defects. The combined polishing processes of laser ablation and tumble finishing have become routine fabrication steps for National Ignition Facility capsule production.« less

In vivo assessment of the structure of skin microcirculation by reflectance confocal-laser-scanning microscopy

NASA Astrophysics Data System (ADS)

Sugata, Keiichi; Osanai, Osamu; Kawada, Hiromitsu

2012-02-01

One of the major roles of the skin microcirculation is to supply oxygen and nutrition to the surrounding tissue. Regardless of the close relationship between the microcirculation and the surrounding tissue, there are few non-invasive methods that can evaluate both the microcirculation and its surrounding tissue at the same site. We visualized microcapillary plexus structures in human skin using in vivo reflectance confocal-laser-scanning microscopy (CLSM), Vivascope 3000® (Lucid Inc., USA) and Image J software (National Institutes of Health, USA) for video image processing. CLSM is a non-invasive technique that can visualize the internal structure of the skin at the cellular level. In addition to internal morphological information such as the extracellular matrix, our method reveals capillary structures up to the depth of the subpapillary plexus at the same site without the need for additional optical systems. Video images at specific depths of the inner forearm skin were recorded. By creating frame-to-frame difference images from the video images using off-line video image processing, we obtained images that emphasize the brightness depending on changes of intensity coming from the movement of blood cells. Merging images from different depths of the skin elucidates the 3-dimensional fine line-structure of the microcirculation. Overall our results show the feasibility of a non-invasive, high-resolution imaging technique to characterize the skin microcirculation and the surrounding tissue.

Real-time confocal laser endomicroscopic evaluation of primary liver cancer based on human liver autofluorescence.

PubMed

Maki, Harufumi; Kawaguchi, Yoshikuni; Arita, Junichi; Akamatsu, Nobuhisa; Kaneko, Junichi; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Harihara, Yasushi; Kokudo, Norihiro

2017-02-01

Confocal laser endomicroscopy (CLE) is available for real-time microscopic examination. This study aims to evaluate the usefulness of intraoperative CLE examination as a modality to evaluate surgical margins in surgery for primary liver cancer. A probe-based CLE system (Cellvizio 100, Mauna Kea Technologies, Paris, France) was used. The subjects comprised seven specimens obtained from six patients with primary liver cancer in November 2015. The probe was manually attached to the surfaces of specimens, and images were collected without external fluorophores. CLE images were compared with hematoxylin and eosin-stained slides. Fluorescence intensity (FI) values of the CLE images were assessed using luminance-analyzing software. CLE examination visualized non-cancerous regions in the background liver as regular structures with high fluorescence because of human liver autofluorescence. Conversely, hepatocellular carcinoma and intrahepatic cholangiocarcinoma were depicted as irregular structures with low fluorescence. The median FI values of the non-cancerous regions and the cancerous regions were 104 (79.8-156) and 74.9 (60.6-106), respectively, and were significantly different (P = 0.031). The probe-based CLE enables real-time differentiation of cancerous regions from non-cancerous tissues in surgical specimens because of human liver autofluorescence. CLE can be used to confirm negative surgical margins in the operating room. J. Surg. Oncol. 2017;115:151-157. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: APPLICATIONS FOR IMAGING MORPHOLOGY AND DEATH IN EMBRYOS AND REPRODUCTIVE TISSUE/ORGANS

EPA Science Inventory

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. It is remarkable that procedures to test the performance of these machines are not done routinely by most investigators and thus many of the machines in the field are working at level...

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

PubMed

Diaspro, A; Corosu, M; Ramoino, P; Robello, M

1999-11-01

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode. Copyright 1999 Wiley-Liss, Inc.

Clinical impact of confocal laser endomicroscopy in the management of gastrointestinal lesions with an uncertain diagnosis.

PubMed

Robles-Medranda, Carlos; Vargas, Maria; Ospina, Jesenia; Puga-Tejada, Miguel; Valero, Manuel; Soria, Miguel; Bravo, Gladys; Robles-Jara, Carlos; Lukashok, Hannah Pitanga

2017-08-16

To evaluate the clinical impact of confocal laser endomicroscopy (CLE) in the diagnosis and management of patients with an uncertain diagnosis. A retrospective chart review was performed. Patients who underwent CLE between November 2013 and October 2015 and exhibited a poor correlation between endoscopic and histological findings were included. Baseline characteristics, indications, previous diagnostic studies, findings at the time of CLE, clinical management and histological results were analyzed. Interventions based on CLE findings were also analyzed. We compared the diagnostic accuracy of CLE and target biopsies of surgical specimens. A total of 144 patients were included. Of these, 51% (74/144) were female. The mean age was 51 years old. In all, 41/144 (28.4%) lesions were neoplastic (13 bile duct, 10 gastric, 8 esophageal, 6 colonic, 1 duodenal, 1 rectal, 1 ampulloma and 1 pancreatic). The sensitivity, specificity, positive predictive value, negative predictive value, and observed agreement when CLE was used to detect N-lesions were 85.37%, 87.38%, 72.92%, 93.75% and 86.81%, respectively. Cohen's Kappa was 69.20%, thus indicating good agreement. Changes in management were observed in 54% of the cases. CLE is a new diagnostic tool that has a significant clinical impact on the diagnosis and treatment of patients with uncertain diagnosis.

Benford's Law based detection of latent fingerprint forgeries on the example of artificial sweat printed fingerprints captured by confocal laser scanning microscopes

NASA Astrophysics Data System (ADS)

Hildebrandt, Mario; Dittmann, Jana

2015-03-01

The possibility of forging latent fingerprints at crime scenes is known for a long time. Ever since it has been stated that an expert is capable of recognizing the presence of multiple identical latent prints as an indicator towards forgeries. With the possibility of printing fingerprint patterns to arbitrary surfaces using affordable ink- jet printers equipped with artificial sweat, it is rather simple to create a multitude of fingerprints with slight variations to avoid raising any suspicion. Such artificially printed fingerprints are often hard to detect during the analysis procedure. Moreover, the visibility of particular detection properties might be decreased depending on the utilized enhancement and acquisition technique. In previous work primarily such detection properties are used in combination with non-destructive high resolution sensory and pattern recognition techniques to detect fingerprint forgeries. In this paper we apply Benford's Law in the spatial domain to differentiate between real latent fingerprints and printed fingerprints. This technique has been successfully applied in media forensics to detect image manipulations. We use the differences between Benford's Law and the distribution of the most significant digit of the intensity and topography data from a confocal laser scanning microscope as features for a pattern recognition based detection of printed fingerprints. Our evaluation based on 3000 printed and 3000 latent print samples shows a very good detection performance of up to 98.85% using WEKA's Bagging classifier in a 10-fold stratified cross-validation.

Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy

PubMed Central

Yokogawa, Hideaki; Kobayashi, Akira; Mori, Natsuko; Sugiyama, Kazuhisa

2015-01-01

Purpose To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Methods Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Results Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Conclusion Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite. PMID:26445524

Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Mori, Natsuko; Sugiyama, Kazuhisa

2015-01-01

To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K.

PubMed

Lange, M; Guénon, S; Lever, F; Kleiner, R; Koelle, D

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4 He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO 3 . The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K

NASA Astrophysics Data System (ADS)

Lange, M.; Guénon, S.; Lever, F.; Kleiner, R.; Koelle, D.

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO3. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

ConfocalGN: A minimalistic confocal image generator

NASA Astrophysics Data System (ADS)

Dmitrieff, Serge; Nédélec, François

Validating image analysis pipelines and training machine-learning segmentation algorithms require images with known features. Synthetic images can be used for this purpose, with the advantage that large reference sets can be produced easily. It is however essential to obtain images that are as realistic as possible in terms of noise and resolution, which is challenging in the field of microscopy. We describe ConfocalGN, a user-friendly software that can generate synthetic microscopy stacks from a ground truth (i.e. the observed object) specified as a 3D bitmap or a list of fluorophore coordinates. This software can analyze a real microscope image stack to set the noise parameters and directly generate new images of the object with noise characteristics similar to that of the sample image. With a minimal input from the user and a modular architecture, ConfocalGN is easily integrated with existing image analysis solutions.

Skin aging: in vivo microscopic assessment of epidermal and dermal changes by means of confocal microscopy.

PubMed

Longo, Caterina; Casari, Alice; Beretti, Francesca; Cesinaro, Anna Maria; Pellacani, Giovanni

2013-03-01

Skin aging is thought to be a complex biological process that is traditionally classified as intrinsic and extrinsic aging. Several clinical score and instrumental devices have been applied to obtain a precise assessment of skin aging. Among them, confocal microscopy has emerged as a new technique capable of assessing cytoarchitectural changes with a nearly histopathologic resolution. We sought to determine the microscopic skin changes occurring on the face in different age groups by means of confocal microscopy. The skin of the cheek in 63 volunteers belonging to distinct age groups was analyzed by confocal microscopy. In 4 cases, routine histopathology was performed on site-matched surplus areas from routine excisions for obtaining a better comparison with confocal findings. Young skin was characterized by regular polygonal keratinocytes and thin reticulated collagen fibers. With aging, more irregularly shaped keratinocytes and areas with unevenly distributed pigmentation and increased compactness of collagen fibers were observed. In the elderly, thinning of the epidermis, marked keratinocyte alterations, and huddles of collagen and curled fibers, corresponding to elastosis, were present. A side-by-side correlation between confocal descriptors and histopathologic aspects has been provided in a few cases. Reticular dermal changes cannot be assessed because of the limited depth laser penetration. Confocal microscopy was successfully applied to identify in vivo skin changes occurring in aged skin at both the epidermal and dermal levels at histopathologic resolution. This offers the possibility to test cosmetic product efficacy and to identify early signs of sun damage. Copyright © 2011 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Evaluation of baseline structural factors for predicting glaucomatous visual-field progression using optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy.

PubMed

Sehi, M; Bhardwaj, N; Chung, Y S; Greenfield, D S

2012-12-01

The objective of this study is to assess whether baseline optic nerve head (ONH) topography and retinal nerve fiber layer thickness (RNFLT) are predictive of glaucomatous visual-field progression in glaucoma suspect (GS) and glaucomatous eyes, and to calculate the level of risk associated with each of these parameters. Participants with ≥28 months of follow-up were recruited from the longitudinal Advanced Imaging for Glaucoma Study. All eyes underwent standard automated perimetry (SAP), confocal scanning laser ophthalmoscopy (CSLO), time-domain optical coherence tomography (TDOCT), and scanning laser polarimetry using enhanced corneal compensation (SLPECC) every 6 months. Visual-field progression was assessed using pointwise linear-regression analysis of SAP sensitivity values (progressor) and defined as significant sensitivity loss of >1 dB/year at ≥2 adjacent test locations in the same hemifield at P<0.01. Cox proportional hazard ratios (HR) were calculated to determine the predictive ability of baseline ONH and RNFL parameters for SAP progression using univariate and multivariate models. Seventy-three eyes of 73 patients (43 GS and 30 glaucoma, mean age 63.2±9.5 years) were enrolled (mean follow-up 51.5±11.3 months). Four of 43 GS (9.3%) and 6 of 30 (20%) glaucomatous eyes demonstrated progression. Mean time to progression was 50.8±11.4 months. Using multivariate models, abnormal CSLO temporal-inferior Moorfields classification (HR=3.76, 95% confidence interval (CI): 1.02-6.80, P=0.04), SLPECC inferior RNFLT (per -1 μm, HR=1.38, 95% CI: 1.02-2.2, P=0.02), and TDOCT inferior RNFLT (per -1 μm, HR=1.11, 95% CI: 1.04-1.2, P=0.001) had significant HRs for SAP progression. Abnormal baseline ONH topography and reduced inferior RNFL are predictive of SAP progression in GS and glaucomatous eyes.

Reflectance confocal microscopy of optical phantoms

PubMed Central

Jacques, Steven L.; Wang, Bo; Samatham, Ravikant

2012-01-01

A reflectance confocal scanning laser microscope (rCSLM) operating at 488-nm wavelength imaged three types of optical phantoms: (1) 100-nm-dia. polystyrene microspheres in gel at 2% volume fraction, (2) solid polyurethane phantoms (INO BiomimicTM), and (3) common reflectance standards (SpectralonTM). The noninvasive method measured the exponential decay of reflected signal as the focus (zf) moved deeper into the material. The two experimental values, the attenuation coefficient μ and the pre-exponential factor ρ, were mapped into the material optical scattering properties, the scattering coefficient μs and the anisotropy of scattering g. Results show that μs varies as 58, 8–24, and 130–200 cm-1 for phantom types (1), (2) and (3), respectively. The g varies as 0.112, 0.53–0.67, and 0.003–0.26, respectively. PMID:22741065

Intensive care unit environmental surfaces are contaminated by multidrug-resistant bacteria in biofilms: combined results of conventional culture, pyrosequencing, scanning electron microscopy, and confocal laser microscopy.

PubMed

Hu, H; Johani, K; Gosbell, I B; Jacombs, A S W; Almatroudi, A; Whiteley, G S; Deva, A K; Jensen, S; Vickery, K

2015-09-01

Hospital-associated infections cause considerable morbidity and mortality, and are expensive to treat. Organisms causing these infections can be sourced from the inanimate environment around a patient. Could the difficulty in eradicating these organisms from the environment be because they reside in dry surface biofilms? The intensive care unit (ICU) of a tertiary referral hospital was decommissioned and the opportunity to destructively sample clinical surfaces was taken in order to investigate whether multidrug-resistant organisms (MDROs) had survived the decommissioning process and whether they were present in biofilms. The ICU had two 'terminal cleans' with 500 ppm free chlorine solution; items from bedding, surrounds, and furnishings were then sampled with cutting implements. Sections were sonicated in tryptone soya broth and inoculated on to chromogenic plates to demonstrate MDROs, which were confirmed with the Vitek2 system. Genomic DNA was extracted directly from ICU samples, and subjected to polymerase chain reaction (PCR) for femA to detect Staphylococcus aureus and the microbiome by bacterial tag-encoded FLX amplicon pyrosequencing. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were performed on environmental samples. Multidrug-resistant bacteria were cultured from 52% (23/44) of samples cultured. S. aureus PCR was positive in 50%. Biofilm was demonstrated in 93% (41/44) of samples by CLSM and/or SEM. Pyrosequencing demonstrated that the biofilms were polymicrobial and contained species that had multidrug-resistant strains. Dry surface biofilms containing MDROs are found on ICU surfaces despite terminal cleaning with chlorine solution. How these arise and how they might be removed requires further study. Copyright © 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Phase Analysis of Laser Direct Etching and Water Assisted Laser Combined Etching of SiC Ceramics

NASA Astrophysics Data System (ADS)

Yuan, Genfu; Cong, Qidong; Zhang, Chen; Xie, Bingbing

2017-12-01

In this study, to discover the etching mechanism of SiC ceramics under laser direct etching and water-jet assisted laser combined etching, the phenomena of substance change on the etched surface were investigated. Also, the rules of substance transfer in etching are discussed. The elemental content change and the phase change of the etching products on the etched surface were analyzed by energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD), respectively. These studies showed a high amount of carbon black on the etched surface, because of the decomposition of SiC ceramics under the high-power-density laser irradiation. SiC decomposed to Si under the laser irradiation, and the subsequent chemical reaction of Si and O2 easily produced SiO2. The SiO2 on the etched surface melted and vaporized, whereas most of SiO2 was removed through splashing, changing the chemical composition of the etched surface. Following the water jet introduction, an increased amount of O existed on the combined etching surface, because the chemical reaction of SiC and H2O easily produced SiO2 under the high-power-density laser irradiation.

Confocal Raman spectroscopy to trace lipstick with their smudges on different surfaces.

PubMed

López-López, Maria; Özbek, Nil; García-Ruiz, Carmen

2014-06-01

Lipsticks are very popular cosmetic products that can be transferred by contact to different surfaces, being important forensic evidence with an intricate analysis if they are found in a crime scene. This study evaluates the use of confocal Raman microscopy at 780 nm excitation wavelength for the nondestructive identification of 49 lipsticks of different brands and colors, overcoming the lipstick fluorescence problem reported by previous works using other laser wavelengths. Although the lipsticks samples showed some fluorescence, this effect was not so intense to completely overwhelm the Raman spectra. Lipsticks smudges on twelve different surfaces commonly stained with these samples were also analyzed. In the case of the surfaces, some of them provided several bands to the smudge spectra compromising the identification of the lipstick. For these samples spectral subtraction of the interfering bands from the surface was performed. Finally, five different red lipsticks with very similar color were measured on different surfaces to evaluate the lipstick traceability with their smudges even on interfering surfaces. Although previous spectral subtraction was needed in some cases, all the smudged were linked to their corresponding lipsticks even when they are smeared on the interfering surfaces. As a consequence, confocal Raman microscopy using the 780 nm excitation laser is presented as a nondestructive powerful tool for the identification of these tricky samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Chondrocytes provide a model for in-situ confocal microscopy and 3D reconstructions

NASA Astrophysics Data System (ADS)

Hirsch, Michelle S.; Svoboda, Kathy K. H.

1994-04-01

Hyaline cartilage is composed of chondrocytes that reside in lacunae surrounded by extracellular matrix molecules. Microscopic and histochemical features of cartilage have been studied with many techniques. Many of these techniques can be time consuming and may alter natural cartilage characteristics. In addition, the orientation and order of sectioned tissue must be maintained to create 3D reconstructions. We show that confocal laser scanning microscopy may replace traditional methods for studying cartilage.

Research on NC laser combined cutting optimization model of sheet metal parts

NASA Astrophysics Data System (ADS)

Wu, Z. Y.; Zhang, Y. L.; Li, L.; Wu, L. H.; Liu, N. B.

2017-09-01

The optimization problem for NC laser combined cutting of sheet metal parts was taken as the research object in this paper. The problem included two contents: combined packing optimization and combined cutting path optimization. In the problem of combined packing optimization, the method of “genetic algorithm + gravity center NFP + geometric transformation” was used to optimize the packing of sheet metal parts. In the problem of combined cutting path optimization, the mathematical model of cutting path optimization was established based on the parts cutting constraint rules of internal contour priority and cross cutting. The model played an important role in the optimization calculation of NC laser combined cutting.

Fabrication of low-loss ridge waveguides in z-cut lithium niobate by combination of ion implantation and UV picosecond laser micromachining

NASA Astrophysics Data System (ADS)

Stolze, M.; Herrmann, T.; L'huillier, J. A.

2016-03-01

Ridge waveguides in ferroelectric materials like LiNbO3 attended great interest for highly efficient integrated optical devices, for instance, electro-optic modulators, frequency converters and ring resonators. The main challenges are the realization of high index barrier towards the substrate and the processing of smooth ridges for minimized scattering losses. For fabricating ridges a variety of techniques, like chemical and wet etching as well as optical grade dicing, have been investigated in detail. Among them, laser micromachining offers a versatile and flexible processing technology, but up to now only a limited side wall roughness has been achieved by this technique. Here we report on laser micromachining of smooth ridges for low-loss optical waveguides in LiNbO3. The ridges with a top width of 7 µm were fabricated in z-cut LiNbO3 by a combination of UV picosecond micromachining and thermal annealing. The laser processing parameters show a strong influence on the achievable sidewall roughness of the ridges and were systematically investigated and optimized. Finally, the surface quality is further improved by an optimized thermal post-processing. The roughness of the ridges were analysed with confocal microscopy and the scattering losses were measured at an optical characterization wavelength of 632.8 nm by using the end-fire coupling method. In these investigations the index barrier was formed by multi-energy low dose oxygen ion implantation technology in a depth of 2.7 μm. With optimized laser processing parameters and thermal post-processing a scattering loss as low as 0.1 dB/cm has been demonstrated.

Effect of calcium on Staphylococcus aureus biofilm architecture: a confocal laser scanning microscopic study.

PubMed

Shukla, Sudhir K; Rao, T Subba

2013-03-01

Bacterial adhesion is a threshold event in the formation of biofilms. Several studies on molecular and biochemical aspects have highlighted that the protein matrix of the biofilm is of interest in developing strategies to combat biofouling. The prevalent role of biofilm associated protein (Bap) of Staphylococcus aureus in early adhesion and the putative presence of Ca(2+) binding EF hand motif in Bap was the motivation for this study. Biofilm assays (S. aureus strains V329 and M556) were done in micro-titer plates and confocal laser scanning microscopy (CLSM) was used to study the biofilm architecture. The results showed that Ca(2+) did not influence planktonic growth of the cultures; however, it modulated the biofilm architecture of S. aureus V329 in a dose dependent manner. Strain M556 was found to be a weak biofilm former and showed no significant change in the presence of Ca(2+). When tested with increasing NaCl concentration, there was no reversal of the Bap-dependent Ca(2+) inhibition of S. aureus V329 biofilm. This indicates that the interaction of Bap and Ca(2+) is not mere electrostatic. CLSM images of V329 biofilm showed reduction in biofilm thickness as well as altered biofilm topography with varying Ca(2+) concentrations. The inhibition effect of Ca(2+) on strain V329 biofilm disappeared in the presence of chelating agent EDTA at a non-inhibiting concentration (0.15 mM). The paper elaborates the role of Ca(2+) in biofilm architecture of S. aureus. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of an integrated automated retinal surgical laser system.

PubMed

Barrett, S F; Wright, C H; Oberg, E D; Rockwell, B A; Cain, C; Rylander, H G; Welch, A J

1996-01-01

Researchers at the University of Texas and the USAF Academy have worked toward the development of a retinal robotic laser system. The overall goal of this ongoing project is to precisely place and control the depth of laser lesions for the treatment of various retinal diseases such as diabetic retinopathy and retinal tears. Separate low speed prototype subsystems have been developed to control lesion depth using lesion reflectance feedback parameters and lesion placement using retinal vessels as tracking landmarks. Both subsystems have been successfully demonstrated in vivo on pigmented rabbits using an argon continuous wave laser. Preliminary testing on rhesus primate subjects have been accomplished with the CW argon laser and also the ultrashort pulse laser. Recent efforts have concentrated on combining the two subsystems into a single prototype capable of simultaneously controlling both lesion depth and placement. We have designated this combined system CALOSOS for Computer Aided Laser Optics System for Ophthalmic Surgery. Several interesting areas of study have developed in integrating the two subsystems: 1) "doughnut" shaped lesions that occur under certain combinations of laser power, spot size, and irradiation time complicating measurements of central lesion reflectance, 2) the optimal retinal field of view (FOV) to achieve both tracking and lesion parameter control, and 3) development of a hybrid analog/digital tracker using confocal reflectometry to achieve retinal tracking speeds of up to 100 dgs. This presentation will discuss these design issues of this clinically significant prototype system. Details of the hybrid prototype system are provided in "Hybrid Eye Tracking for Computer-Aided Retinal Surgery" at this conference. The paper will close with remaining technical hurdles to clear prior to testing the full-up clinical prototype system.

Association of fluorescein anterior corneal mosaic and corneal K-structures by in vivo laser confocal microscopy in patients with keratoconus.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Mori, Natsuko; Masaki, Toshinori; Sugiyama, Kazuhisa

2017-01-01

To report the in vivo laser confocal microscopy findings of corneas with keratoconus, with special attention to abnormality of Bowman's layer and sub-Bowman's fibrous structures (Kobayashi-structures [K-structures]). Sixteen keratoconic eyes in 8 consecutive patients with keratoconus (4 males, 4 females, mean age, 41.1 years) were included in this study. Slit-lamp biomicroscopic photos were taken with or without fluorescein staining. The existence of anterior corneal mosaic (ACM) after eyelid rubbing under fluorescein staining was documented. In vivo laser confocal microscopic examinations were performed for all patients in both the central cone and the peripheral cornea to examine the existence of K-structures. According to the Amsler-Krumeich scale, the eyes were graded as follows: stage 1 (n=3), stage 2 (n=1), stage 3 (n=1), and stage 4 (n=11). ACM was observed in 7 eyes (61.1%) in the cone area and 16 eyes (100%) in the peripheral cornea among all keratoconic eyes enrolled in this study. In addition, K-structures were observed in the 7 eyes (61.1%) and 16 eyes (100%) in the peripheral cornea among all keratoconic eyes. The presence of the K-structures was completely matched (100%) with the presence of ACM in both the central cone and the peripheral cornea. In 11 eyes with stage 4 keratoconus, ACM and K-structure was absent in 9 eyes (81.8%) in the cone area. On the contrary, in 5 eyes with mild-to-moderate keratoconus (grade 1 to 3), ACM and K-structure was present in all eyes (100%) in the cone area. The absent ratio of ACM and K-structures in the cone area was significantly higher in stage 4 severe keratoconus compared to mild-to-moderate keratoconus (grade 1 to 3) (Fisher, P =0.005). The existence of ACM and K-structures in both the central cone and the peripheral cornea showed perfect accord in patients with keratoconus, indicating a strong association of ACM and K-structures in patients with keratoconus. With the progress of the keratoconus, it seemed that

Association of fluorescein anterior corneal mosaic and corneal K-structures by in vivo laser confocal microscopy in patients with keratoconus

PubMed Central

Kobayashi, Akira; Yokogawa, Hideaki; Mori, Natsuko; Masaki, Toshinori; Sugiyama, Kazuhisa

2017-01-01

Objective To report the in vivo laser confocal microscopy findings of corneas with keratoconus, with special attention to abnormality of Bowman’s layer and sub-Bowman’s fibrous structures (Kobayashi-structures [K-structures]). Methods Sixteen keratoconic eyes in 8 consecutive patients with keratoconus (4 males, 4 females, mean age, 41.1 years) were included in this study. Slit-lamp biomicroscopic photos were taken with or without fluorescein staining. The existence of anterior corneal mosaic (ACM) after eyelid rubbing under fluorescein staining was documented. In vivo laser confocal microscopic examinations were performed for all patients in both the central cone and the peripheral cornea to examine the existence of K-structures. Results According to the Amsler–Krumeich scale, the eyes were graded as follows: stage 1 (n=3), stage 2 (n=1), stage 3 (n=1), and stage 4 (n=11). ACM was observed in 7 eyes (61.1%) in the cone area and 16 eyes (100%) in the peripheral cornea among all keratoconic eyes enrolled in this study. In addition, K-structures were observed in the 7 eyes (61.1%) and 16 eyes (100%) in the peripheral cornea among all keratoconic eyes. The presence of the K-structures was completely matched (100%) with the presence of ACM in both the central cone and the peripheral cornea. In 11 eyes with stage 4 keratoconus, ACM and K-structure was absent in 9 eyes (81.8%) in the cone area. On the contrary, in 5 eyes with mild-to-moderate keratoconus (grade 1 to 3), ACM and K-structure was present in all eyes (100%) in the cone area. The absent ratio of ACM and K-structures in the cone area was significantly higher in stage 4 severe keratoconus compared to mild-to-moderate keratoconus (grade 1 to 3) (Fisher, P=0.005). Conclusion The existence of ACM and K-structures in both the central cone and the peripheral cornea showed perfect accord in patients with keratoconus, indicating a strong association of ACM and K-structures in patients with keratoconus. With the

Image-guided intraocular injection using multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy in rodent ophthalmological models

NASA Astrophysics Data System (ADS)

Terrones, Benjamin D.; Benavides, Oscar R.; Leeburg, Kelsey C.; Mehanathan, Sankarathi B.; Levine, Edward M.; Tao, Yuankai K.

2018-02-01

Intraocular injections are routinely performed for delivery of anti-VEGF and anti-inflammatory therapies in humans. While these injections are also performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the injection location and volume are not well-controlled and reproducible. We overcome limitations of conventional injections methods by developing a multimodality, long working distance, non-contact optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (cSLO) system for retinal imaging before and after injections. Our OCT+cSLO system combines a custom-built spectraldomain OCT engine (875+/-85 nm) with 125 kHz line-rate with a modified commercial cSLO with a maximum frame-rate of 30 fps (512 x 512 pix.). The system was designed for an overlapping OCT+cSLO field-of-view of 1.1 mm with a 7.76 mm working distance to the pupil. cSLO excitation light sources and filters were optimized for simultaneous GFP and tdTomato imaging. Lateral resolution was 3.02 µm for OCT and 2.74 μm for cSLO. Intravitreal injections of 5%, 10%, and 20% intralipid with Alex Fluor 488 were manually injected intraocularly in C57BL/6 mice. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. OCT enables quantitative analysis of injection location and volumes whereas complementary cSLO improves specificity for identifying fluorescently labeled injected compounds and transgenic cells. The long working distance of our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections and may be applied for imaging of ophthalmic surgical dynamics and real-time image-guided injections.

Multimodal ophthalmic imaging using swept source spectrally encoded scanning laser ophthalmoscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

Malone, Joseph D.; El-Haddad, Mohamed T.; Tye, Logan A.; Majeau, Lucas; Godbout, Nicolas; Rollins, Andrew M.; Boudoux, Caroline; Tao, Yuankai K.

2016-03-01

Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) benefit clinical diagnostic imaging in ophthalmology by enabling in vivo noninvasive en face and volumetric visualization of retinal structures, respectively. Spectrally encoding methods enable confocal imaging through fiber optics and reduces system complexity. Previous applications in ophthalmic imaging include spectrally encoded confocal scanning laser ophthalmoscopy (SECSLO) and a combined SECSLO-OCT system for image guidance, tracking, and registration. However, spectrally encoded imaging suffers from speckle noise because each spectrally encoded channel is effectively monochromatic. Here, we demonstrate in vivo human retinal imaging using a swept source spectrally encoded scanning laser ophthalmoscope and OCT (SSSESLO- OCT) at 1060 nm. SS-SESLO-OCT uses a shared 100 kHz Axsun swept source, shared scanner and imaging optics, and are detected simultaneously on a shared, dual channel high-speed digitizer. SESLO illumination and detection was performed using the single mode core and multimode inner cladding of a double clad fiber coupler, respectively, to preserve lateral resolution while improving collection efficiency and reducing speckle contrast at the expense of confocality. Concurrent en face SESLO and cross-sectional OCT images were acquired with 1376 x 500 pixels at 200 frames-per-second. Our system design is compact and uses a shared light source, imaging optics, and digitizer, which reduces overall system complexity and ensures inherent co-registration between SESLO and OCT FOVs. En face SESLO images acquired concurrent with OCT cross-sections enables lateral motion tracking and three-dimensional volume registration with broad applications in multivolume OCT averaging, image mosaicking, and intraoperative instrument tracking.

Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy.

PubMed

Keevil, C W

2003-01-01

Knowledge of biofilm structure and function has changed significantly in the last few years due to advances in light microscopy. One pertinent example is the use of scanning confocal laser microscopy (SCLM) to visualise corrosion pits caused by the biofilm mosaic footprint on corroding metal surfaces. Nevertheless, SCLM has some limitations as to its widespread use, including cost, inability to observe motile bacteria and eukaryotic grazers within biofilms, and difficulty to scan a curved surface. By contrast, episcopic differential interference contrast (EDIC) microscopy has provided a rapid, real time analysis of biofilms on opaque, curved, natural or man-made surfaces without the need for cover slips and oil. EDIC, coupled with epi-fluorescence (EDIC/EF), microscopy has been used successfully to visualise the 3-D biofilm structure, physiological niches, protozoal grazing and iron biomineralization, and the location of specific pathogens such as Legionella pneumophila, Campylobacter jejuni and Cryptosporidium parvum. These species were identified using gold nanoparticles or fluorophores coupled to monoclonal antibodies or 16S rRNA probes, respectively. Among its many potential uses, the EDIC technique will provide a rapid procedure to facilitate the calibration of the modern generation of biofilm-sensing electrodes.

Role of laser fluence in protein synthesis of cultured DRG neurons following low-level laser irradiation

NASA Astrophysics Data System (ADS)

Zheng, Liqin; Qiu, Caimin; Wang, Yuhua; Zeng, Yixiu; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

2014-11-01

Low-level lasers have been used to relieve pain in clinical for many years. But the mechanism is not fully clear. In animal models, nitric oxide (NO) has been reported involving in the transmission and modulation of nociceptive signals. So the objective of this study was to establish whether low-level laser with different fluence could stimulate the production of nitric oxide synthese (NOS), which produces NO in cultured primary dorsal root ganglion neurons (DRG neurons). The primary DRG neurons were isolated from healthy Sprague Dawley rats (8-12 weeks of age) and spread on 35 mm culture dishes specially used for confocal microscopy. 24 hours after spreading, cells were irradiated with 658 nm laser for two consecutive days at the energy density of 20, 40, 60 and 80 mJ·cm-2 respectively. Control groups were not exposed to the laser, but were kept under the same conditions as the irradiated ones. The synthesis of NOS after laser irradiation was detected by immunofluorescence assay, and the changes of NOS were evaluated using confocal microscopy and Image J software. The results showed that all the laser fluence could promote the production of NOS in DRG neurons, especially the 60 mJ·cm-2 . These results demonstrated that low-level laser irradiation could modify protein synthesis in a dose- or fluence- dependent manner, and indicated that low-level laser irradiation might achieve the analgesic effect through modulation of NO production.

New Laser System For Combined Monitoring And Treatment Of Neonatal Hyperbilirubinemia

NASA Astrophysics Data System (ADS)

Hamza, Mostafa; Hamza, Mohammad

1989-09-01

Laser photoradiation therapy for neonatal hyperbilirubinemia is a breakthrough in the management of neonatal jaundice. In this paper the authors present a new laser system that provides combined monitoring and therapy for neonatal hyperbilirubinemia. The new system incorporates tunable laser sources that can be operated at selected wavelengths to achieve both transcutaneous differential absorption measurements of bilirubin concentration in addition to laser photoradiation therapy. The new laser system can allow the treating physician to avoid over or under treatment of jaundiced neonates by the control of serum bilirubin from a critically high level to a reasonably safe level.

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

Comparison of solar and laser macula retinal injury using scanning laser ophthalmoscopy spectral imaging

NASA Astrophysics Data System (ADS)

Zwick, Harry; Gagliano, Donald A.; Stuck, Bruce E.; Lund, David J.

1994-07-01

Both solar and laser sources may induce punctate foveal retinal damage. Unprotected viewing of the sun or bright blue sky represent potential solar radiation causes of photic maculopathy that may induce punctate foveal damage. Laser induced macular retinal damage is another more recent kind of photic maculopathy. Most documented cases of laser photic maculopathy have involved acute laser exposure generally from Q-switched visible or nonvisible near IR laser systems. In our comparison of these types of photic maculopathies, we have employed conventional as well as spectral and confocal scanning laser ophthalomoscopy to evaluate the depth of the photic maculopathy. Functionally, we have observed a tritan color vision loss present in nearly all photic maculopathies.

Noninvasive in situ observation of the crystallization kinetics of biological macromolecules by confocal laser scanning microscopy.

PubMed

Mühlig, P; Klupsch, Th; Kaulmann, U; Hilgenfeld, R

2003-04-01

High-resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein crystal growth kinetics. Because the resolution of CLSM is not diffraction-limited by the object, it is possible to visualize, under certain conditions, objects in molecular dimensions. A modified batch technique is applied which allows the growth kinetics of sufficiently small crystallites fixed at the lower side of a cover glass, within a hanging drop, to be studied in reflected light near the total reflection angle. A gap, or cavity, filled with solution is formed between the cover glass and the upper crystal face, which acts to fix small crystallites by hydrodynamic friction forces. The cavity height enables the propagation of molecular steps across the upper crystal face without constraint, so that the propagation velocity and geometrical parameters can be measured by CLSM. The layer growth kinetics of monoclinic crystallites of a long-acting insulin derivative (Insulin Glargine) is investigated. For a twofold supersaturation of the solution, the growth is governed by 2D nucleation at the edges of the crystallites followed by a spreading of molecular steps. The layer growth kinetics are well fitted by the simple cubic kinetic lattice model. We find that only about one of a thousand solute (protein) molecules which push a kink place due to their Brownian motion becomes really incorporated into the growing crystal.

Clinical impact of confocal laser endomicroscopy in the management of gastrointestinal lesions with an uncertain diagnosis

PubMed Central

Robles-Medranda, Carlos; Vargas, Maria; Ospina, Jesenia; Puga-Tejada, Miguel; Valero, Manuel; Soria, Miguel; Bravo, Gladys; Robles-Jara, Carlos; Lukashok, Hannah Pitanga

2017-01-01

AIM To evaluate the clinical impact of confocal laser endomicroscopy (CLE) in the diagnosis and management of patients with an uncertain diagnosis. METHODS A retrospective chart review was performed. Patients who underwent CLE between November 2013 and October 2015 and exhibited a poor correlation between endoscopic and histological findings were included. Baseline characteristics, indications, previous diagnostic studies, findings at the time of CLE, clinical management and histological results were analyzed. Interventions based on CLE findings were also analyzed. We compared the diagnostic accuracy of CLE and target biopsies of surgical specimens. RESULTS A total of 144 patients were included. Of these, 51% (74/144) were female. The mean age was 51 years old. In all, 41/144 (28.4%) lesions were neoplastic (13 bile duct, 10 gastric, 8 esophageal, 6 colonic, 1 duodenal, 1 rectal, 1 ampulloma and 1 pancreatic). The sensitivity, specificity, positive predictive value, negative predictive value, and observed agreement when CLE was used to detect N-lesions were 85.37%, 87.38%, 72.92%, 93.75% and 86.81%, respectively. Cohen’s Kappa was 69.20%, thus indicating good agreement. Changes in management were observed in 54% of the cases. CONCLUSION CLE is a new diagnostic tool that has a significant clinical impact on the diagnosis and treatment of patients with uncertain diagnosis. PMID:28874959

High-Temperature Confocal Laser Scanning Microscopy Studies of Ferrite Formation in Inclusion-Engineered Steels: A Review

NASA Astrophysics Data System (ADS)

Mu, Wangzhong; Hedström, Peter; Shibata, Hiroyuki; Jönsson, Pär G.; Nakajima, Keiji

2018-05-01

The concepts of oxide metallurgy and inclusion engineering can be utilized to improve the properties of low-alloy steels. These concepts aim at controlling the formation of intragranular ferrite (IGF), often a desirable microstructure providing good mechanical properties without the need for expensive alloying elements. IGF formation is stimulated to occur at non-metallic inclusions and form an arrangement of fine, interlocking ferrite grains. A method that has contributed significantly to investigations in this field lately is high-temperature confocal laser scanning microscopy (HT-CLSM). HT-CLSM is suited for in situ studies of inclusion behavior in liquid steel and phase transformations in solid-state steel, where in particular, displacive phase transformations can be studied, since they provide sufficient topographic contrast. The purpose of the present report is to provide a brief review of the state of the art of HT-CLSM and its application for in situ observations of ferrite formation in inclusion-engineered steels. The scientific literature in this field is surveyed and supplemented by new work to reveal the capability of HT-CLSM as well as to discuss the effect of factors such as cooling rate and parent grain size on IGF formation and growth kinetics. The report concludes with an outlook on the opportunities and challenges of HT-CLSM for applications in oxide metallurgy.

Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

PubMed

Cushway, T R; Lanzetta, M; Cox, G; Trickett, R; Owen, E R

1996-01-01

Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy.

16 W output power by high-efficient spectral beam combining of DBR-tapered diode lasers.

PubMed

Müller, André; Vijayakumar, Deepak; Jensen, Ole Bjarlin; Hasler, Karl-Heinz; Sumpf, Bernd; Erbert, Götz; Andersen, Peter E; Petersen, Paul Michael

2011-01-17

Up to 16 W output power has been obtained using spectral beam combining of two 1063 nm DBR-tapered diode lasers. Using a reflecting volume Bragg grating, a combining efficiency as high as 93.7% is achieved, resulting in a single beam with high spatial coherence. The result represents the highest output power achieved by spectral beam combining of two single element tapered diode lasers. Since spectral beam combining does not affect beam propagation parameters, M2-values of 1.8 (fast axis) and 3.3 (slow axis) match the M2-values of the laser with lowest spatial coherence. The principle of spectral beam combining used in our experiments can be expanded to combine more than two tapered diode lasers and hence it is expected that the output power may be increased even further in the future.

Central serous chorioretinopathy fundus autofluorescence comparison with two different confocal scanning laser ophthalmoscopes.

PubMed

Nam, Ki Tae; Yun, Cheol Min; Kim, Jee Taek; Yang, Kyung-Sook; Kim, Hyun Joo; Kim, Seong-Woo; Oh, Jaeryung; Huh, Kuhl

2015-12-01

To compare the lesion characteristics of two different types of confocal scanning laser ophthalmoscopy (cSLO) autofluorescence (AF) images in central serous chorioretinopathy (CSC). The study included 63 eyes of 61 patients; 63 pairs of fundus autofluorescence (FAF) images were compared before CSC resolution in 63 eyes, FAF images of 31 eyes were also compared after CSC resolution. The lesion characteristics (brightness and composite pattern) were compared between Heidelberg Retina Angiograph 2 (HRA2; Heidelberg Engineering, Germany) and Optomap Tx (Optomap; Optos, Scotland) FAF images. The lesion composite pattern was categorized as diffuse or granular. Diffuse AF was defined as homogenously increased or decreased AF, and granular AF was defined as dot-like, coarse changes in AF. The mean disease duration and subretinal fluid (SRF) height in the spectral domain optical coherence tomography were compared according to the FAF image characteristics. Lesion brightness before CSC resolution was hypo-AF in 48 eyes (76.2 %), hyper-AF in three (4.8 %), and mixed-AF in 12 (19.0 %) in HRA2 FAF images. In comparison, nine (14.3 %) images were hypo-AF, 44 (69.8 %) were hyper-AF, and 10 (15.9 %) were mixed-AF in Optomap FAF images (P < 0.0001). There was no significant difference in lesion composite pattern between the two FAF image wavelengths. Patients with lesions that were hyper-AF in Optomap FAF and hypo-AF in HRA2 FAF had a shorter disease duration and greater SRF height (1 month, 281 um) than those who were hyper-AF in both Optomap and HRA2 images (26 months, 153 um; P = 0.004, 0.001). The two types of FAF images of CSC showed different lesion brightness before and after CSC resolution but demonstrated similar lesion composite patterns.

Coherent combining of high brightness tapered lasers in master oscillator power amplifier configuration

NASA Astrophysics Data System (ADS)

Albrodt, P.; Hanna, M.; Moron, F.; Decker, J.; Winterfeldt, M.; Blume, G.; Erbert, G.; Crump, P.; Georges, P.; Lucas-Leclin, G.

2018-02-01

Improved diode laser beam combining techniques are in strong demand for applications in material processing. Coherent beam combining (CBC) is the only combining approach that has the potential to maintain or even improve all laser properties, and thus has high potential for future systems. As part of our ongoing studies into CBC of diode lasers, we present recent progress in the coherent superposition of high-power single-pass tapered laser amplifiers. The amplifiers are seeded by a DFB laser at λ = 976 nm, where the seed is injected into a laterally single-mode ridge-waveguide input section. The phase pistons on each beam are actively controlled by varying the current in the ridge section of each amplifier, using a sequential hill-climbing algorithm, resulting in a combined beam with power fluctuations of below 1%. The currents into the tapered sections of the amplifiers are separately controlled, and remain constant. In contrast to our previous studies, we favour a limited number of individual high-power amplifiers, in order to preserve a high extracted power per emitter in a simple, low-loss coupling arrangement. Specifically, a multi-arm interferometer architecture with only three devices is used, constructed using 6 mm-long tapered amplifiers, mounted junction up on C-mounts, to allow separate contact to single mode and amplifier sections. A maximum coherently combined power of 12.9 W is demonstrated in a nearly diffraction-limited beam, corresponding to a 65% combining efficiency, with power mainly limited by the intrinsic beam quality of the amplifiers. Further increased combined power is currently sought.

Multiple beam interference confocal microscopy: a tool for morphological investigation of living cells and tissues

NASA Astrophysics Data System (ADS)

Joshi, Narahari V.; Medina, Honorio

2000-05-01

Multiple beam interference system is used in conjunction with a conventional scanning confocal microscope to examine the morphology and construction of 3D images of Histolytic Ameba and parasite Candida Albicans. The present combination permits to adjoin advantages of both systems, namely the vertical high contrast and optical sectioning. The interference pattern obtained from a multiple internal reflection of a simple, sandwiched between the glass plate and the cover plate, was focussed on an objective of a scanning confocal microscope. According to optical path differences, morphological details were revealed. The combined features, namely improved resolution in z axis, originated from the interference pattern and the optical sectioning of the confocal scanning system, enhance the resolution and contrast dramatically. These features permitted to obtain unprecedented images of Histolytic Ameba and parasite Candida Albicans. Because of the improved contrast, several details like double wall structure of candida, internal structure of ameba are clearly visible.

A Clinical and Confocal Microscopic Comparison of Transepithelial PRK and LASEK for Myopia

PubMed Central

Korkmaz, Safak; Bilgihan, Kamil; Sul, Sabahattin; Hondur, Ahmet

2014-01-01

Purpose. To compare the clinical and confocal microscopic results of transepithelial PRK versus LASEK for correction of myopia. Materials and Methods. Twelve patients with myopia received transepithelial PRK in one eye and LASEK in the other. In transepithelial PRK-treated eyes, the corneal epithelium was removed with 40 microns of excimer laser ablation and in LASEK-treated eyes with 25-second application of 18% ethanol. Time to epithelial healing, ocular discomfort, uncorrected and best corrected visual acuities, manifest refraction, haze, greyscale value, and keratocyte apoptosis in confocal microscopy were recorded. Results. The mean time to epithelial healing was significantly longer after LASEK (4.00 ± 0.43 versus 3.17 ± 0.6 days). On day 1, ocular discomfort was significantly higher after transepithelial PRK. The grade of haze, keratocyte apoptosis, and greyscale value in confocal microscopy were significantly higher in transepithelial PRK-treated eyes at 1 month. All transepithelial PRK- and LASEK-treated eyes achieved 20/25 or better UCVA and were within ±1.00 D of emmetropia at final visits. Conclusions. Both transepithelial PRK and LASEK offer effective correction of myopia at 1 year. However, LASEK appeared to induce less discomfort and less intense wound healing in the early postoperative period. PMID:25120924

A Clinical and Confocal Microscopic Comparison of Transepithelial PRK and LASEK for Myopia.

PubMed

Korkmaz, Safak; Bilgihan, Kamil; Sul, Sabahattin; Hondur, Ahmet

2014-01-01

Purpose. To compare the clinical and confocal microscopic results of transepithelial PRK versus LASEK for correction of myopia. Materials and Methods. Twelve patients with myopia received transepithelial PRK in one eye and LASEK in the other. In transepithelial PRK-treated eyes, the corneal epithelium was removed with 40 microns of excimer laser ablation and in LASEK-treated eyes with 25-second application of 18% ethanol. Time to epithelial healing, ocular discomfort, uncorrected and best corrected visual acuities, manifest refraction, haze, greyscale value, and keratocyte apoptosis in confocal microscopy were recorded. Results. The mean time to epithelial healing was significantly longer after LASEK (4.00 ± 0.43 versus 3.17 ± 0.6 days). On day 1, ocular discomfort was significantly higher after transepithelial PRK. The grade of haze, keratocyte apoptosis, and greyscale value in confocal microscopy were significantly higher in transepithelial PRK-treated eyes at 1 month. All transepithelial PRK- and LASEK-treated eyes achieved 20/25 or better UCVA and were within ±1.00 D of emmetropia at final visits. Conclusions. Both transepithelial PRK and LASEK offer effective correction of myopia at 1 year. However, LASEK appeared to induce less discomfort and less intense wound healing in the early postoperative period.

A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy.

PubMed

Verveer, P. J; Gemkow, M. J; Jovin, T. M

1999-01-01

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.

[Results of the ocular hypertension treatment study and the confocal scanning laser ophthalmoscopy ancillary study and evaluation of the heidelberg retina tomograph].

PubMed

Klatt, K; Schmidt, E; Scheuerle, A F

2008-04-01

The Ocular Hypertension Treatment Study (OHTS) has shown that analyzing changes of the optic disc configuration is superior to evaluating visual field findings for the early detection of primary open angle glaucoma. The Confocal Scanning Laser Ophthalmoscopy Ancillary Study (CSLO) is the first study to reveal that certain topographic baseline measurements of the optic disc are significantly associated with the development of primary open angle glaucoma in patients with ocular hypertension. An abnormally increased "mean height contour" value proved to be the individual parameter connected with the highest risk. The reliability of the Moorfields Regression Analysis of certain individual sectors during early detection of a primary angle glaucoma is higher than that of the global measurement. The temporal superior and inferior as well as the nasal inferior sectors have the highest positive predictive values and the largest risks in both univariate and multivariate analysis.

Phase conjugation of high energy lasers.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bliss, David E; Valley, Michael T.; Atherton, Briggs W.

2013-01-01

In this report we explore claims that phase conjugation of high energy lasers by stimulated Brillouin scattering (SBS) can compensate optical aberrations associated with severely distorted laser amplifier media and aberrations induced by the atmosphere. The SBS media tested was a gas cell pressurized up to 300 psi with SF6 or Xe or both. The laser was a 10 Hz, 3J, Q-switched Nd:YAG with 25 ns wide pulses. Atmospheric aberrations were created with space heaters, helium jets and phase plates designed with a Kolmogorov turbulence spectrum characterized by a Fried parameter, ro , ranging from 0.6 6.0 mm. Phase conjugatemore » tests in the laboratory were conducted without amplification. For the strongest aberrations, D/ro ~ 20, created by combining the space heaters with the phase plate, the Strehl ratio was degraded by a factor of ~50. Phase conjugation in SF6 restored the peak focusable intensity to about 30% of the original laser. Phase conjugate tests at the outdoor laser range were conducted with laser amplifiers providing gain in combination with the SBS cell. A large 600,000 BTU kerosene space heater was used to create turbulence along the beam path. An atmospheric structure factor of Cn2 = 5x10-13 m2/3 caused the illumination beam to expand to a diameter 250mm and overfill the receiver. The phase conjugate amplified return could successfully be targeted back onto glints 5mm in diameter. Use of a lenslet arrays to lower the peak focusable intensity in the SBS cell failed to produce a useful phase conjugate beam; The Strehl ratio was degraded with multiple random lobes instead of a single focus. I will review literature results which show how multiple beams can be coherently combined by SBS when a confocal reflecting geometry is used to focus the laser in the SBS cell.« less

Fractional Carbon Dioxide Laser and its Combination with Subcision in Improving Atrophic Acne Scars.

PubMed

Nilforoushzadeh, Mohammad Ali; Faghihi, Gita; Jaffary, Fariba; Haftbaradaran, Elaheh; Hoseini, Sayed Mohsen; Mazaheri, Nafiseh

2017-01-01

Acne is a very common skin disease in which scars are seen in 95% of the patients. Although numerous treatments have been recommended, researchers are still searching for a single modality to treat the complication due to its variety in shape and depth. We compared the effects of fractional carbon dioxide (CO 2 ) laser alone and in combination with subcision in the treatment of atrophic acne scars. This clinical trial study was performed in Skin Diseases and Leishmaniasis Research Center (Isfahan, Iran) during 2011-2012. Eligible patients with atrophic acne scars were treated with fractional CO 2 laser alone (five sessions with 3-week interval) on the right side of the face and fractional CO 2 laser plus subcision (one session using both with four sessions of fractional CO 2 laser, with 3-week interval) on the left side. The subjects were visited 1, 2, and 6 months after the treatment. Patient satisfaction rate was analyzed using SPSS 20 software. The average of recovery rate was 54.7% using the combination method and 43.0% using laser alone ( P < 0.001). The mean patient satisfaction was significantly higher with the combination method than laser alone (6.6 ± 1.2 vs. 5.2 ± 1.8; P < 0.001). Bruising was only seen with the combination method and lasted for 1 week in 57.0% and for 2 weeks in 43.0%. Erythema was seen in both methods. Postinflammatory pigmentation and hyperpigmentation were associated with combination method. No persistent side effects were seen after 6 months. Using a combination of subcision and laser had suitable results regarding scar recovery and satisfaction rate.

Numerical study of a confocal ultrasonic setup for creation of cavitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lafond, Maxime, E-mail: maxime.lafond@inserm.fr; Chavrier, Françoise; Prieur, Fabrice

2015-10-28

Acoustic cavitation is used for various therapeutic applications such as local enhancement of drug delivery, histotripsy or hyperthermia. One of the utmost important parameter for cavitation creation is the rarefaction pressure. The typical magnitude of the rarefaction pressure required to initiate cavitation from gas dissolved in tissue is beyond the range of the megapascal. Because nonlinear effects need to be taken into account, a numerical simulator based on the Westervelt equation was used to study the pressure waveform and the acoustic field generated by a setup for creation of cavitation consisting of two high intensity focused ultrasound transducers mounted confocally.more » At constant acoustic power, simulations with only one and both transducers from the confocal setup showed that the distortion of the pressure waveform due to the combined effects of nonlinearity and diffraction is less pronounced when both confocal transducers are used. Consequently, the confocal setup generates a greater peak negative pressure at focus which is more favorable for cavitation initiation. Comparison between the confocal setup and a single transducer with the same total emitting surface puts in evidence the role of the spatial separation of the two beams. Furthermore, it has been previously shown that the location of the peak negative pressure created by a single transducer shifts from focus towards the transducers in the presence of nonlinear effects. The simulator was used to study a configuration where the acoustical axes of transducers intersect on the peak negative pressure instead of the geometrical focus. For a representative confocal setup, namely moderate nonlinear effects, a 2% increase of the peak negative pressure and 8% decrease of the peak positive pressure resulted from this configuration. These differences tend to increase by increasing nonlinear effects. Although the optimal position of the transducers varies with the nonlinear regimen, the intersection

Living Matter Observations with a Novel Hyperspectral Supercontinuum Confocal Microscope for VIS to Near-IR Reflectance Spectroscopy

PubMed Central

Bertani, Francesca R.; Ferrari, Luisa; Mussi, Valentina; Botti, Elisabetta; Costanzo, Antonio; Selci, Stefano

2013-01-01

A broad range hyper-spectroscopic microscope fed by a supercontinuum laser source and equipped with an almost achromatic optical layout is illustrated with detailed explanations of the design, implementation and data. The real novelty of this instrument, a confocal spectroscopic microscope capable of recording high resolution reflectance data in the VIS-IR spectral range from about 500 nm to 2.5 μm wavelengths, is the possibility of acquiring spectral data at every physical point as defined by lateral coordinates, X and Y, as well as at a depth coordinate, Z, as obtained by the confocal optical sectioning advantage. With this apparatus we collect each single scanning point as a whole spectrum by combining two linear spectral detector arrays, one CCD for the visible range, and one InGaAs infrared array, simultaneously available at the sensor output channel of the home made instrument. This microscope has been developed for biomedical analysis of human skin and other similar applications. Results are shown illustrating the technical performances of the instrument and the capability in extracting information about the composition and the structure of different parts or compartments in biological samples as well as in solid statematter. A complete spectroscopic fingerprinting of samples at microscopic level is shown possible by using statistical analysis on raw data or analytical reflectance models based on Abelés matrix transfer methods. PMID:24233077

Side-pumping combiner for high-power fiber laser based on tandem pumping

NASA Astrophysics Data System (ADS)

Gu, Yanran; Lei, Chengmin; Liu, Jun; Li, Ruixian; Liu, Le; Xiao, Hu; Chen, Zilun

2017-11-01

We investigate a (2+1)×1 side-pumping combiner numerically and experimentally for high-power fiber laser based on tandem pumping for the first time. The influence of taper ratio and launch mode on the 1018-nm pump coupling efficiency and the leakage power into the coating of the signal fiber (LPC) is analyzed numerically. A side-pumping combiner is developed successfully by tapered-fused splicing technique based on the numerical analysis, consisting of two pump fibers (220/242 μm, NA=0.22) and a signal fiber (40/400 μm, NA=0.06/0.46). The total 1018-nm pump efficiency of the combiner is 98.1%, and the signal light insertion loss is <3%. The results show that, compared with laser diodes pumping, the combiner appears to have a better LPC performance and power handling capability when using 1018-nm fiber as the pump light. Meanwhile, an all-fiber MOPA laser based on tandem pumping with 1080-nm output of 2533 W and the slope efficiency of 82.8% is achieved based on the home-made combiner.

Comparison of Q-switched Nd:YAG laser alone versus its combination with ultrapulse CO2 laser for the treatment of black tattoo.

PubMed

Vanarase, Mithila; Gautam, Ram Krishan; Arora, Pooja; Bajaj, Sonali; Meena, Neha; Khurana, Ananta

2017-10-01

Q-switched lasers are conventionally used for the treatment of black tattoo. However, they require multiple sittings, and the response may be slow due to competing epidermal pigment in dark skin. To compare the efficacy of Q-switched Nd:YAG laser alone with its combination with ultrapulse CO 2 for the removal of black tattoo. Sixty patients with black tattoo were randomized into two groups viz., group A and group B. Group A was treated with QS Nd:YAG laser (1064 nm) alone, and group B received combination of ablative ultrapulse CO 2 followed by fixed-dose QS Nd:YAG laser (1064 nm), at 6-week interval for a maximum of 6 sittings. After each sitting, 3 independent physicians noted percentage of improvement that was evaluated using visual analogue scale (VAS) and grading system for tattoo ink lightening (TIL). Combination laser (group B) showed statistically significant improvement in mean VAS score in the last 2 noted visits as compared to 1st session (p < 0.007, p < 0.001) and TIL mean score in last three noted visits as compared to 1st session (p < 0.008, p < 0.020, and p < 0.004). There was no statistically significant difference in the side effect profile of both the groups. For refractory professional tattoos, combination of ultrapulse CO 2 laser and QS Nd:YAG laser is superior to QS Nd:YAG laser alone.

Histopathological confirmation of similar intramucosal distribution of fluorescein in both intravenous administration and local mucosal application for probe-based confocal laser endomicroscopy of the normal stomach.

PubMed

Nonaka, Kouichi; Ohata, Ken; Ban, Shinichi; Ichihara, Shin; Takasugi, Rumi; Minato, Yohei; Tashima, Tomoaki; Matsuyama, Yasushi; Takita, Maiko; Matsuhashi, Nobuyuki; Neumann, Helmut

2015-12-16

Probe-based confocal laser endomicroscopy (pCLE) is capable of acquiring in vivo magnified cross-section images of the gastric mucosa. Intravenous injection of fluorescein sodium is used for confocal imaging. However, it is still under debate if local administration of the dye to the mucosa is also effective for confocal imaging as it is not yet clear if topical application also reveals the intramucosal distribution of fluorescein. The objective of this study was to evaluate the intramucosal distribution of fluorescein sodium after topical application and to compare the distribution to the conventional intravenous injection used for confocal imaging. pCLE of the stomach uninfected with Helicobacter pylori was performed in a healthy male employing intravenous administration and local mucosal application of fluorescein. The mucosa of the lower gastric body was biopsied 1 min and 5 min after intravenous administration or local mucosal application of fluorescein, and the distribution of fluorescein in the biopsy samples was examined histologically. Green fluorescence was already observed in the cytoplasm of fundic glandular cells in the biopsied deep mucosa 1 min after local mucosal application of fluorescein. It was also observed in the foveolar lumen and inter-foveolar lamina propria, although it was noted at only a few sites. In the tissue biopsied 5 min after the local mucosal application of fluorescein, green fluorescence was more frequently noted in the cytoplasm of fundic glandular cells than in that 1 min after the local mucosal application of fluorescein, although obvious green fluorescence was not identified in the foveolar lumen or inter-foveolar lamina propria. The distribution of intravenously administered fluorescein in the cytoplasm of fundic glandular cells was also clearly observed similarly to that after local mucosal application of fluorescein. Green fluorescence in more cells was observed in many cells 5 min after intravenous administration compared

MEMS-based handheld confocal microscope for in-vivo skin imaging

PubMed Central

Arrasmith, Christopher L.; Dickensheets, David L.; Mahadevan-Jansen, Anita

2010-01-01

This paper describes a handheld laser scanning confocal microscope for skin microscopy. Beam scanning is accomplished with an electromagnetic MEMS bi-axial micromirror developed for pico projector applications, providing 800x600 (SVGA) resolution at 56 frames per second. The design uses commercial objective lenses with an optional hemisphere front lens, operating with a range of numerical aperture from NA=0.35 to NA=1.1 and corresponding diagonal field of view ranging from 653 μm to 216 μm. Using NA=1.1 and a laser wavelength of 830 nm we measured the axial response to be 1.14 μm full width at half maximum, with a corresponding 10%-90% lateral edge response of 0.39 μm. Image examples showing both epidermal and dermal features including capillary blood flow are provided. These images represent the highest resolution and frame rate yet achieved for tissue imaging with a MEMS bi-axial scan mirror. PMID:20389391

Automatic classification of small bowel mucosa alterations in celiac disease for confocal laser endomicroscopy

NASA Astrophysics Data System (ADS)

Boschetto, Davide; Di Claudio, Gianluca; Mirzaei, Hadis; Leong, Rupert; Grisan, Enrico

2016-03-01

Celiac disease (CD) is an immune-mediated enteropathy triggered by exposure to gluten and similar proteins, affecting genetically susceptible persons, increasing their risk of different complications. Small bowels mucosa damage due to CD involves various degrees of endoscopically relevant lesions, which are not easily recognized: their overall sensitivity and positive predictive values are poor even when zoom-endoscopy is used. Confocal Laser Endomicroscopy (CLE) allows skilled and trained experts to qualitative evaluate mucosa alteration such as a decrease in goblet cells density, presence of villous atrophy or crypt hypertrophy. We present a method for automatically classifying CLE images into three different classes: normal regions, villous atrophy and crypt hypertrophy. This classification is performed after a features selection process, in which four features are extracted from each image, through the application of homomorphic filtering and border identification through Canny and Sobel operators. Three different classifiers have been tested on a dataset of 67 different images labeled by experts in three classes (normal, VA and CH): linear approach, Naive-Bayes quadratic approach and a standard quadratic analysis, all validated with a ten-fold cross validation. Linear classification achieves 82.09% accuracy (class accuracies: 90.32% for normal villi, 82.35% for VA and 68.42% for CH, sensitivity: 0.68, specificity 1.00), Naive Bayes analysis returns 83.58% accuracy (90.32% for normal villi, 70.59% for VA and 84.21% for CH, sensitivity: 0.84 specificity: 0.92), while the quadratic analysis achieves a final accuracy of 94.03% (96.77% accuracy for normal villi, 94.12% for VA and 89.47% for CH, sensitivity: 0.89, specificity: 0.98).

Combination of fractional erbium-glass laser and topical therapy in melasma resistant to triple-combination cream.

PubMed

Tourlaki, Athanasia; Galimberti, Michela Gianna; Pellacani, Giovanni; Bencini, Pier Luca

2014-06-01

Melasma is a common melanosis often difficult to treat. The aim of this paper was to report on the safety and efficacy of non-ablative fractional photothermolysis combined with the use of triple-combination cream (TCC) on a large population with melasma resistant (i.e., with no complete/near-complete clearing) to TCC alone. Seventy-six patients with resistant melasma underwent a combined treatment protocol. The protocol consisted of a TCC (hydroquinone 4%, retinoic acid 0.03%, hydrocortisone butyrate 0.1%) applied daily for 10 days followed by four laser treatments performed in 3-week intervals with a fractional 1540-nm erbium-glass laser. During these intervals, and for 3 months after the last laser session, TCC was also applied daily following a "pulse-therapy" scheme. Improvement was assessed by the melasma-area-and-severity-index (MASI) score. At 1 month, marked (>75%) and moderate (51-75%) clearing of melasma were observed in 46 of 76 (67.1%) and 12 of 76 (21%) cases, respectively. At 6 months, we noticed a marked improvement in 16 of 76 (21.1%) and no improvement in 33 of 76 (43.4%) patients. Our study proposes the combination of NFP/TCC as a useful therapy for patients with melasma resistant to TCC alone, but it shows that its long-term efficacy is limited.

Three-dimensional confocal microscopy of the living cornea and ocular lens

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-07-01

The three-dimensional reconstruction of the optic zone of the cornea and the ocular crystalline lens has been accomplished using confocal microscopy and volume rendering computer techniques. A laser scanning confocal microscope was used in the reflected light mode to obtain the two-dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with a 488 nm wavelength. The microscope objective was a Leitz X25, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133 three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their 'beaded' cell borders, basal lamina, nerve plexus, nerve fibers, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in- situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers. The three-dimensional data sets of the cornea and the ocular lens were reconstructed in the computer using volume rendering techniques. Stereo pairs were also created of the two- dimensional ocular images for visualization. The stack of two-dimensional images was reconstructed into a three-dimensional object using volume rendering techniques. This demonstration of the three-dimensional visualization of the intact, enucleated eye provides an important step toward quantitative three-dimensional morphometry of the eye. The important aspects of three-dimensional reconstruction are discussed.

Volumetry of human taste buds using laser scanning microscopy.

PubMed

Just, T; Srur, E; Stachs, O; Pau, H W

2009-10-01

In vivo laser scanning confocal microscopy is a relatively new, non-invasive method for assessment of oral cavity epithelia. The penetration depth of approximately 200-400 microm allows visualisation of fungiform papillae and their taste buds. This paper describes the technique of in vivo volumetry of human taste buds. Confocal laser scanning microscopy used a diode laser at 670 nm for illumination. Digital laser scanning confocal microscopy equipment consisted of the Heidelberg Retina Tomograph HRTII and the Rostock Cornea Module. Volume scans of fungiform papillae were used for three-dimensional reconstruction of the taste bud. This technique supplied information on taste bud structure and enabled measurement and calculation of taste bud volume. Volumetric data from a 23-year-old man over a nine-day period showed only a small deviation in values. After three to four weeks, phenomenological changes in taste bud structures were found (i.e. a significant increase in volume, followed by disappearance of the taste bud and appearance of a new taste bud). The data obtained indicate the potential application of this non-invasive imaging modality: to evaluate variation of taste bud volume in human fungiform papillae with ageing; to study the effects of chorda tympani nerve transection on taste bud volume; and to demonstrate recovery of taste buds in patients with a severed chorda tympani nerve who show recovery of gustatory sensibility after surgery.

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

Chromatic confocal microscope using hybrid aspheric diffractive lenses

NASA Astrophysics Data System (ADS)

Rayer, Mathieu; Mansfield, Daniel

2014-05-01

A chromatic confocal microscope is a single point non-contact distance measurement sensor. For three decades the vast majority of the chromatic confocal microscope use refractive-based lenses to code the measurement axis chromatically. However, such an approach is limiting the range of applications. In this paper the performance of refractive, diffractive and Hybrid aspheric diffractive are compared. Hybrid aspheric diffractive lenses combine the low geometric aberration of a diffractive lens with the high optical power of an aspheric lens. Hybrid aspheric diffractive lenses can reduce the number of elements in an imaging system significantly or create large hyper- chromatic lenses for sensing applications. In addition, diffractive lenses can improve the resolution and the dynamic range of a chromatic confocal microscope. However, to be suitable for commercial applications, the diffractive optical power must be significant. Therefore, manufacturing such lenses is a challenge. We show in this paper how a theoretical manufacturing model can demonstrate that the hybrid aspheric diffractive configuration with the best performances is achieved by step diffractive surface. The high optical quality of step diffractive surface is then demonstrated experimentally. Publisher's Note: This paper, originally published on 5/10/14, was replaced with a corrected/revised version on 5/19/14. If you downloaded the original PDF but are unable to access the revision, please contact SPIE Digital Library Customer Service for assistance.

4Pi-confocal microscopy of live cells

NASA Astrophysics Data System (ADS)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

Realistic representation of Bacillus subtilis biofilms architecture using combined microscopy (CLSM, ESEM and FESEM).

PubMed

Bridier, A; Meylheuc, T; Briandet, R

2013-05-01

In this contribution, we used a set of microscopic techniques including confocal laser scanning microscopy (CLSM), environmental scanning electron microscopy (ESEM) and field emission scanning electron microscopy (FESEM) to analyze the three-dimensional spatial arrangement of cells and their surrounding matrix in Bacillus subtilis biofilm. The combination of the different techniques enabled a deeper and realistic deciphering of biofilm architecture by providing the opportunity to overcome the limits of each single technique. Copyright © 2013 Elsevier Ltd. All rights reserved.

Two-photon confocal microscopy in wound healing

NASA Astrophysics Data System (ADS)

Navarro, Fernando A.; So, Peter T. C.; Driessen, Antoine; Kropf, Nina; Park, Christine S.; Huertas, Juan C.; Lee, Hoon B.; Orgill, Dennis P.

2001-04-01

Advances in histopathology and immunohistochemistry have allowed for precise microanatomic detail of tissues. Two Photon Confocal Microscopy (TPCM) is a new technology useful in non-destructive analysis of tissue. Laser light excites the natural florophores, NAD(P)H and NADP+ and the scattering patterns of the emitted light are analyzed to reconstruct microanatomic features. Guinea pig skin was studied using TPCM and skin preparation methods including chemical depilation and tape striping. Results of TPCM were compared with conventional hematoxylin and eosin microscopy. Two-dimensional images were rendered from the three dimensional reconstructions. Images of deeper layers including basal cells and the dermo-epidermal junction improved after removing the stratum corneum with chemical depilation or tape stripping. TCPM allows good resolution of corneocytes, basal cells and collagen fibers and shows promise as a non-destructive method to study wound healing.

Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts

NASA Astrophysics Data System (ADS)

Cang, Hu; Wong, Chung M.; Xu, C. Shan; Rizvi, Abbas H.; Yang, Haw

2006-05-01

We present an apparatus that noninvasively tracks a moving nanoparticle in three dimensions while providing concurrent sequential spectroscopic measurements. The design, based on confocal microscopy, uses a near-infrared laser and a dark-field condenser for illumination of a gold nanoparticle. By monitoring the scattered light from the nanoparticle and using a piezoelectric stage, the system was able to continuously bring the diffusive particle in a glycerol/water solution back to the focal volume with spatial resolution and response time of less than 210nm and a millisecond, respectively.

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.

Development of high damage threshold multilayer thin film beam combiner for laser application

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nand, Mangla, E-mail: mnand@rrcat.gov.in; Babita,; Jena, S.

2016-05-23

A polarized wavelength multiplexer with high laser induced damage threshold has been developed to combine two laser beam of high peak power in the visible region. The present wavelength multiplexer is a multilayer thin film device deposited by reactive electron beam evaporation. The developed device is capable of combining two p-polarized laser beams of peak power density of 1.7 GW/cm{sup 2} at an angle of incidence of 45°. High transmission (T> 90%) in high pass region and high reflection (R> 99%) in stop band region have been achieved.

Development of high damage threshold multilayer thin film beam combiner for laser application

NASA Astrophysics Data System (ADS)

Nand, Mangla; Babita, Jena, S.; Tokas, R. B.; Rajput, P.; Mukharjee, C.; Thakur, S.; Jha, S. N.; Sahoo, N. K.

2016-05-01

A polarized wavelength multiplexer with high laser induced damage threshold has been developed to combine two laser beam of high peak power in the visible region. The present wavelength multiplexer is a multilayer thin film device deposited by reactive electron beam evaporation. The developed device is capable of combining two p-polarized laser beams of peak power density of 1.7 GW/cm2 at an angle of incidence of 45°. High transmission (T> 90%) in high pass region and high reflection (R> 99%) in stop band region have been achieved.

Evaluation of baseline structural factors for predicting glaucomatous visual-field progression using optical coherence tomography, scanning laser polarimetry and confocal scanning laser ophthalmoscopy

PubMed Central

Sehi, M; Bhardwaj, N; Chung, Y S; Greenfield, D S

2012-01-01

Purpose The objective of this study is to assess whether baseline optic nerve head (ONH) topography and retinal nerve fiber layer thickness (RNFLT) are predictive of glaucomatous visual-field progression in glaucoma suspect (GS) and glaucomatous eyes, and to calculate the level of risk associated with each of these parameters. Methods Participants with ≥28 months of follow-up were recruited from the longitudinal Advanced Imaging for Glaucoma Study. All eyes underwent standard automated perimetry (SAP), confocal scanning laser ophthalmoscopy (CSLO), time-domain optical coherence tomography (TDOCT), and scanning laser polarimetry using enhanced corneal compensation (SLPECC) every 6 months. Visual-field progression was assessed using pointwise linear-regression analysis of SAP sensitivity values (progressor) and defined as significant sensitivity loss of >1 dB/year at ≥2 adjacent test locations in the same hemifield at P<0.01. Cox proportional hazard ratios (HR) were calculated to determine the predictive ability of baseline ONH and RNFL parameters for SAP progression using univariate and multivariate models. Results Seventy-three eyes of 73 patients (43 GS and 30 glaucoma, mean age 63.2±9.5 years) were enrolled (mean follow-up 51.5±11.3 months). Four of 43 GS (9.3%) and 6 of 30 (20%) glaucomatous eyes demonstrated progression. Mean time to progression was 50.8±11.4 months. Using multivariate models, abnormal CSLO temporal-inferior Moorfields classification (HR=3.76, 95% confidence interval (CI): 1.02–6.80, P=0.04), SLPECC inferior RNFLT (per −1 μm, HR=1.38, 95% CI: 1.02–2.2, P=0.02), and TDOCT inferior RNFLT (per −1 μm, HR=1.11, 95% CI: 1.04–1.2, P=0.001) had significant HRs for SAP progression. Conclusion Abnormal baseline ONH topography and reduced inferior RNFL are predictive of SAP progression in GS and glaucomatous eyes. PMID:23060026

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

Comparison possibilities of ultrasound and its combination with laser in surgery and therapy

NASA Astrophysics Data System (ADS)

Zharov, Vladimir P.; Menyaev, Yulian A.; Kabisov, Ruslan K.; Alkov, Sergey V.; Nesterov, A. V.; Loshchilov, Vladimir I.; Suen, James Y.

2000-05-01

This article presents the further developments of combined laser-ultrasound medical technologies with paying attention the possibility ultrasound in surgery and therapy. The analyses of main effects at the low frequency ultrasonic treatment of biotissues including cavitation, acoustic streams, acoustic pressure, mechanical influence etc are analyzed. The main promising areas of application of low frequency ultrasound are considered including bactericidal treatment of infections wounds, spray treatment of wounds in head and neck surgery, tumor treatment etc. In particular the clinical result of using ultrasonic devices based on imposing ultrasonic oscillations in a range of 22-66 kHz on a cutting instrument with a special form, radiation intensity up to 10 W/cm2 and oscillation amplitude up to 40-60 micrometers with respect to oncology for halt bleeding from a tumor, liquidating pain, acoustic denervation are presented. Some limitation of medical application of ultrasound are discussed and perspective combination with laser for increasing efficiency of new combined technologies are found. Among them: combination photodynamic therapy and ultrasonic treatment of tumors, laser-ultrasonic treatment of infections wounds including using spray, laser-ultrasonic drug delivery. The preliminary result of experimental study of some of above-mentioned technologies are presented.

Gold nanorods for cell imaging with confocal reflectance microscopy and two-photon fluorescence microscopy

NASA Astrophysics Data System (ADS)

Chen, Ji-Yao; Wang, Pei-Nan

2010-02-01

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

1-kilowatt CW all-fiber laser oscillator pumped with wavelength-beam-combined diode stacks.

PubMed

Xiao, Y; Brunet, F; Kanskar, M; Faucher, M; Wetter, A; Holehouse, N

2012-01-30

We have demonstrated a monolithic cladding-pumped ytterbium-doped single all-fiber laser oscillator generating 1 kW of CW signal power at 1080 nm with 71% slope efficiency and near diffraction-limited beam quality. Fiber components were highly integrated on "spliceless" passive fibers to promote laser efficiency and alleviate non-linear effects. The laser was pumped through a 7:1 pump combiner with seven 200-W 91x nm fiber-pigtailed wavelength-beam-combined diode-stack modules. The signal power of such a single all-fiber laser oscillator showed no evidence of roll-over, and the highest output was limited only by available pump power.

Surface Modification of ICF Target Capsules by Pulsed Laser Ablation

DOE PAGES

Carlson, Lane C.; Johnson, Michael A.; Bunn, Thomas L.

2016-06-30

Topographical modifications of spherical surfaces are imprinted on National Ignition Facility (NIF) target capsules by extending the capabilities of a recently developed full surface (4π) laser ablation and mapping apparatus. The laser ablation method combines the precision, energy density and long reach of a focused laser beam to pre-impose sinusoidal modulations on the outside surface of High Density Carbon (HDC) capsules and the inside surface of Glow Discharge Polymer (GDP) capsules. Sinusoidal modulations described in this paper have sub-micron to 10’s of microns vertical scale and wavelengths as small as 30 μm and as large as 200 μm. The modulatedmore » patterns are created by rastering a focused laser fired at discrete capsule surface locations for a specified number of pulses. The computer program developed to create these raster patterns uses inputs such as laser beam intensity profile, the material removal function, the starting surface figure and the desired surface figure. The patterns are optimized to minimize surface roughness. Lastly, in this paper, simulated surfaces are compared with actual ablated surfaces measured using confocal microscopy.« less

Optical feedback effects on terahertz quantum cascade lasers: modelling and applications

NASA Astrophysics Data System (ADS)

Rakić, Aleksandar D.; Lim, Yah Leng; Taimre, Thomas; Agnew, Gary; Qi, Xiaoqiong; Bertling, Karl; Han, She; Wilson, Stephen J.; Kundu, Iman; Grier, Andrew; Ikonić, Zoran; Valavanis, Alexander; Demić, Aleksandar; Keeley, James; Li, Lianhe H.; Linfield, Edmund H.; Davies, A. Giles; Harrison, Paul; Ferguson, Blake; Walker, Graeme; Prow, Tarl; Indjin, Dragan; Soyer, H. Peter

2016-11-01

Terahertz (THz) quantum cascade lasers (QCLs) are compact sources of radiation in the 1-5 THz range with significant potential for applications in sensing and imaging. Laser feedback interferometry (LFI) with THz QCLs is a technique utilizing the sensitivity of the QCL to the radiation reflected back into the laser cavity from an external target. We will discuss modelling techniques and explore the applications of LFI in biological tissue imaging and will show that the confocal nature of the QCL in LFI systems, with their innate capacity for depth sectioning, makes them suitable for skin diagnostics with the well-known advantages of more conventional confocal microscopes. A demonstration of discrimination of neoplasia from healthy tissue using a THz, LFI-based system in the context of melanoma is presented using a transgenic mouse model.

Confocal microlaparoscope for imaging the fallopian tube

NASA Astrophysics Data System (ADS)

Wu, Tzu-Yu; Rouse, Andrew R.; Chambers, Setsuko K.; Hatch, Kenneth D.; Gmitro, Arthur F.

2014-11-01

Recent evidence suggests that ovarian cancer can originate in the fallopian tube. Unlike many other cancers, poor access to the ovary and fallopian tubes has limited the ability to study the progression of this deadly disease and to diagnosis it during the early stage when it is most amenable to therapy. A rigid confocal microlaparoscope system designed to image the epithelial surface of the ovary in vivo was previously reported. A new confocal microlaparoscope with an articulating distal tip has been developed to enable in vivo access to human fallopian tubes. The new microlaparoscope is compatible with 5-mm trocars and includes a 2.2-mm-diameter articulating distal tip consisting of a bare fiber bundle and an automated dye delivery system for fluorescence confocal imaging. This small articulating device should enable the confocal microlaparoscope to image early stage ovarian cancer arising inside the fallopian tube. Ex vivo images of animal tissue and human fallopian tube using the new articulating device are presented along with in vivo imaging results using the rigid confocal microlaparoscope system.

High harmonic terahertz confocal gyrotron with nonuniform electron beam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Wenjie; Guan, Xiaotong; Yan, Yang

2016-01-15

The harmonic confocal gyrotron with nonuniform electron beam is proposed in this paper in order to develop compact and high power terahertz radiation source. A 0.56 THz third harmonic confocal gyrotron with a dual arc section nonuniform electron beam has been designed and investigated. The studies show that confocal cavity has extremely low mode density, and has great advantage to operate at high harmonic. Nonuniform electron beam is an approach to improve output power and interaction efficiency of confocal gyrotron. A dual arc beam magnetron injection gun for designed confocal gyrotron has been developed and presented in this paper.

Confocal Fluorescence Microscopy of Mung Beanleaves

NASA Astrophysics Data System (ADS)

Chen, Zhiwei; Liu, Dongwu

Recently, confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. The light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, which is one of the critical features of the confocal microscope. In present studies, the probes acridine orange (AO) and rhodamine-123 were used to research stoma and mitochondria of mung bean leaves, respectively. The results indicated that the stomatal guard cells and mitochondria were clearly seen in epidermic tissue of mung bean leaves. Taken together, it is a good method to research plant cells with confocal microscope and fluorescence probes.

COMBINE*: An integrated opto-mechanical tool for laser performance modeling

NASA Astrophysics Data System (ADS)

Rehak, M.; Di Nicola, J. M.

2015-02-01

Accurate modeling of thermal, mechanical and optical processes is important for achieving reliable, high-performance high energy lasers such as those at the National Ignition Facility [1] (NIF). The need for this capability is even more critical for high average power, high repetition rate applications. Modeling the effects of stresses and temperature fields on optical properties allows for optimal design of optical components and more generally of the architecture of the laser system itself. Stresses change the indices of refractions and induce inhomogeneities and anisotropy. We present a modern, integrated analysis tool that efficiently produces reliable results that are used in our laser propagation tools such as VBL [5]. COMBINE is built on and supplants the existing legacy tools developed for the previous generations of lasers at LLNL but also uses commercially available mechanical finite element codes ANSYS or COMSOL (including computational fluid dynamics). The COMBINE code computes birefringence and wave front distortions due to mechanical stresses on lenses and slabs of arbitrary geometry. The stresses calculated typically originate from mounting support, vacuum load, gravity, heat absorption and/or attending cooling. Of particular importance are the depolarization and detuning effects of nonlinear crystals due to thermal loading. Results are given in the form of Jones matrices, depolarization maps and wave front distributions. An incremental evaluation of Jones matrices and ray propagation in a 3D mesh with a stress and temperature field is performed. Wavefront and depolarization maps are available at the optical aperture and at slices within the optical element. The suite is validated, user friendly, supported, documented and amenable to collaborative development. * COMBINE stands for Code for Opto-Mechanical Birefringence Integrated Numerical Evaluations.

Comparison of a long-pulse Nd:YAG laser and a combined 585/1,064-nm laser for the treatment of acne scars: a randomized split-face clinical study.

PubMed

Min, Seong U K; Choi, Yu Sung; Lee, Dong Hun; Yoon, Mi Young; Suh, Dae Hun

2009-11-01

Nonablative laser is gaining popularity because of the low risk of complications, especially in patients with darker skin. To compare the efficacy and safety of a long-pulse neodymium-doped yttrium aluminium garnet (Nd:YAG) laser and a combined 585/1,064-nm laser for the treatment of acne scars. Nineteen patients with mild to moderate atrophic acne scars received four long-pulse Nd:YAG laser or combined 585/1,064-nm laser treatment sessions at fortnightly intervals. Treatments were administered randomly in a split-face manner. Acne scars showed mild to moderate improvement, with significant Echelle d'évaluation clinique des cicatrices d'acné (ECCA) score reductions, after both treatments. Although intermodality differences were not significant, combined 585/1,064-nm laser was more effective for deep boxcar scars. In patients with combined 585/1,064-nm laser-treated sides that improved more than long-pulse Nd:YAG laser-treated sides, ECCA scores were significantly lower for combined 585/1,064-nm laser treatment. Histologic evaluations revealed significantly greater collagen deposition, although there was no significant difference between the two modalities. Patient satisfaction scores concurred with physicians' evaluations. Both lasers ameliorated acne scarring with minimal downtime. In light of this finding, optimal outcomes might be achieved when laser treatment types are chosen after considering individual scar type and response.

High incidence of rainbow glare after femtosecond laser assisted-LASIK using the upgraded FS200 femtosecond laser.

PubMed

Zhang, Yu; Chen, Yue-Guo

2018-03-05

To compare the incidence of rainbow glare (RG) after femtosecond laser assisted-LASIK (FS-LASIK) using the upgraded FS200 femtosecond laser with different flap cut parameter settings. A consecutive series of 129 patients (255 eyes) who underwent FS-LASIK for correcting myopia and/or astigmatism using upgraded WaveLight FS200 femtosecond laser with the original settings was included in group A. Another consecutive series of 129 patients (255 eyes) who underwent FS-LASIK using upgraded WaveLight FS200 femtosecond laser with flap cut parameter settings changed (decreased pulse energy, spot and line separation) was included in group B. The incidence and fading time of RG, confocal microscopic image and postoperative clinical results were compared between the two groups. There were no differences between the two groups in age, baseline refraction, excimer laser ablation depth, postoperative uncorrected visual acuity and refraction. The incidence rate of RG in group A (35/255, 13.73%) was significantly higher than that in group B (4/255, 1.57%) (P < 0.05). The median fading time was 3 months in group A and 1 month in group B (P > 0.05).The confocal microscopic images showed wider laser spot spacing in group A than group B. The incidence of RG was significantly correlated with age and grouping (P < 0.05). The upgraded FS200 femtosecond laser with original flap cut parameter settings could increase the incidence of RG. The narrower grating size and lower pulse energy could ameliorate this side effect.

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.

Laser beam combining and cleanup by stimulated Brillouin scattering in a multimode optical fiber.

PubMed

Rodgers, B C; Russell, T H; Roh, W B

1999-08-15

A new technique for combining low-power laser beams has been demonstrated by use of semiconductor diode lasers. The technique, which is appropriate for any single-longitudinal-mode laser, is based on stimulated Brillouin scattering (SBS) in long multimode optical fibers. It produces a clean Gaussian-like beam that corresponds to the fundamental fiber mode, irrespective of the profile of the pump. Coherent as well as incoherent combining was demonstrated, and conversion slope efficiencies as high as 67% and 83% were shown to be achievable for the single-pass and ring-cavity SBS geometries, respectively.

Confocal Endomicroscopy: Instrumentation and Medical Applications

PubMed Central

Jabbour, Joey M.; Saldua, Meagan A.; Bixler, Joel N.; Maitland, Kristen C.

2013-01-01

Advances in fiber optic technology and miniaturized optics and mechanics have propelled confocal endomicroscopy into the clinical realm. This high resolution, non-invasive imaging technology provides the ability to microscopically evaluate cellular and sub-cellular features in tissue in vivo by optical sectioning. Because many cancers originate in epithelial tissues accessible by endoscopes, confocal endomicroscopy has been explored to detect regions of possible neoplasia at an earlier stage by imaging morphological features in vivo that are significant in histopathologic evaluation. This technique allows real-time assessment of tissue which may improve diagnostic yield by guiding biopsy. Research and development continues to reduce the overall size of the imaging probe, increase the image acquisition speed, and improve resolution and field of view of confocal endomicroscopes. Technical advances will continue to enable application to less accessible organs and more complex systems in the body. Lateral and axial resolutions down to 0.5 μm and 3 μm, respectively, field of view as large as 800×450 μm, and objective lens and total probe outer diameters down to 350 μm and 1.25 mm, respectively, have been achieved. We provide a review of the historical developments of confocal imaging in vivo, the evolution of endomicroscope instrumentation, and the medical applications of confocal endomicroscopy. PMID:21994069

Evaluation of confocal microscopy system performance.

PubMed

Zucker, R M; Price, O

2001-08-01

The confocal laser scanning microscope (CLSM) has been used by scientists to visualize three-dimensional (3D) biological samples. Although this system involves lasers, electronics, optics, and microscopes, there are few published tests that can be used to assess the performance of this equipment. Usually the CLSM is assessed by subjectively evaluating a biological/histological test slide for image quality. Although there is a use for the test slide, there are many other components in the CLSM that need to be assessed. It would be useful if tests existed that produced reference values for machine performance. The aim of this research was to develop quality assurance tests to ensure that the CLSM was stable while delivering reproducible intensity measurements with excellent image quality. Our ultimate research objective was to quantify fluorescence using a CLSM. To achieve this goal, it is essential that the CLSM be stable while delivering known parameters of performance. Using Leica TCS-SP1 and TCS-4D systems, a number of tests have been devised to evaluate equipment performance. Tests measuring dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, laser stability, photomultiplier tube (PMT) reliability, and system noise were either incorporated from the literature or derived in our laboratory to measure performance. These tests are also applicable to other manufacturer's systems with minor modifications. A preliminary report from our laboratory has addressed a number of the QA issues necessary to achieve CLSM performance. This report extends our initial work on the evaluation of CLSM system performance. Tests that were described previously have been modified and new tests involved in laser stability and sensitivity are described. The QA tests on the CLSM measured laser power, PMT function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance

A total internal reflection-fluorescence correlation spectroscopy setup with pulsed diode laser excitation

NASA Astrophysics Data System (ADS)

Weger, Lukas; Hoffmann-Jacobsen, Kerstin

2017-09-01

Fluorescence correlation spectroscopy (FCS) measures fluctuations in a (sub-)femtoliter volume to analyze the diffusive behavior of fluorescent particles. This highly sensitive method has proven to be useful for the analysis of dynamic biological systems as well as in chemistry, physics, and material sciences. It is routinely performed with commercial fluorescence microscopes, which provide a confined observation volume by the confocal technique. The evanescent wave of total internal reflectance (TIR) is used in home-built systems to permit a surface sensitive FCS analysis. We present a combined confocal and TIR-FCS setup which uses economic low-power pulsed diode lasers for excitation. Excitation and detection are coupled to time-correlated photon counting hardware. This allows simultaneous fluorescence lifetime and FCS measurements in a surface-sensitive mode. Moreover, the setup supports fluorescence lifetime correlation spectroscopy at surfaces. The excitation can be easily switched between TIR and epi-illumination to compare the surface properties with those in liquid bulk. The capabilities of the presented setup are demonstrated by measuring the diffusion coefficients of a free dye molecule, a labeled polyethylene glycol, and a fluorescent nanoparticle in confocal as well as in TIR-FCS.

An Automatic Procedure for Combining Digital Images and Laser Scanner Data

NASA Astrophysics Data System (ADS)

Moussa, W.; Abdel-Wahab, M.; Fritsch, D.

2012-07-01

Besides improving both the geometry and the visual quality of the model, the integration of close-range photogrammetry and terrestrial laser scanning techniques directs at filling gaps in laser scanner point clouds to avoid modeling errors, reconstructing more details in higher resolution and recovering simple structures with less geometric details. Thus, within this paper a flexible approach for the automatic combination of digital images and laser scanner data is presented. Our approach comprises two methods for data fusion. The first method starts by a marker-free registration of digital images based on a point-based environment model (PEM) of a scene which stores the 3D laser scanner point clouds associated with intensity and RGB values. The PEM allows the extraction of accurate control information for the direct computation of absolute camera orientations with redundant information by means of accurate space resection methods. In order to use the computed relations between the digital images and the laser scanner data, an extended Helmert (seven-parameter) transformation is introduced and its parameters are estimated. Precedent to that, in the second method, the local relative orientation parameters of the camera images are calculated by means of an optimized Structure and Motion (SaM) reconstruction method. Then, using the determined transformation parameters results in having absolute oriented images in relation to the laser scanner data. With the resulting absolute orientations we have employed robust dense image reconstruction algorithms to create oriented dense image point clouds, which are automatically combined with the laser scanner data to form a complete detailed representation of a scene. Examples of different data sets are shown and experimental results demonstrate the effectiveness of the presented procedures.

Combined pulsed dye and CO2 lasers in the treatment of angiolymphoid hyperplasia with eosinophilia.

PubMed

Sagi, Lior; Halachmi, Shlomit; Levi, Assi; Amitai, Dan Ben; Enk, Claes D; Lapidoth, Moshe

2016-08-01

Angiolymphoid hyperplasia with eosinophilia (ALHE) is an uncommon dermatosis of unknown etiology that manifests as characteristic red nodules and papules with a predilection for the scalp and periauricular region. Treatment is required for both esthetic and functional reasons, as lesions may ulcerate and bleed. Many treatment approaches have been reported, including excision, systemic medical approaches, topical or intralesional therapies, and non-invasive modalities including cryotherapy, electrosurgery, and laser. Treatments have exhibited variable efficacy, and the recurrence rate is 100 %. We report the combination of pulsed dye laser and CO2 laser in the treatment of ALHE in 14 patients. All patients exhibited clinical response after a mean of 2.4 ± 0.4 treatment sessions. The clinical efficacy of the combined treatment, together with its well-tolerated nature, render the use of pulsed dye laser in combination with CO2 laser, a viable treatment for debulking ALHE lesions. Ongoing maintenance treatments are needed to due to the high degree of relapse.

Clinical efficacy of flumetasone/salicylic acid ointment combined with 308-nm excimer laser for treatment of psoriasis vulgaris.

PubMed

Dong, Jie; He, Yanling; Zhang, Xiuying; Wang, Yixuan; Tian, Yongjing; Wang, Jie

2012-06-01

To compare the clinical efficacy and safety of combining flumetasone ointment with 308-nm excimer laser therapy vs. 308-nm excimer laser monotherapy for the treatment of psoriasis vulgaris. Forty patients with psoriasis vulgaris were recruited; 20 were treated with flumetasone ointment plus 308-nm excimer laser therapy, and the other 20 received only excimer laser monotherapy. The flumetasone ointment was applied topically twice a day, and laser treatments were scheduled twice weekly for a total of 10 treatments. Clinical efficacy was evaluated in a blinded manner by two independent physicians using photographs taken before and after treatment. Of the 40 patients who received and completed the entire course of therapy, the psoriasis area and severity index score was improved by 82.51 ± 11.24% and 72.01 ± 20.94% in the combination group and laser group, respectively (P > 0.05), and the average cumulative dose was 5.06 ± 2.20 j/cm(2) in the combination group and 7.75 ± 2.25 j/cm(2) in the laser-only group, respectively (P < 0.05). The clinical data suggest that combination treatment using flumetasone ointment and a 308-nm excimer laser is superior to laser monotherapy for treatment of psoriasis vulgaris. The combination therapy can increase effectiveness and decrease the total laser dose, thus potentially reducing side effects. © 2012 John Wiley & Sons A/S.

Crystallization Behavior of the CaO-Al2O3-MgO System Studied with a Confocal Laser Scanning Microscope

NASA Astrophysics Data System (ADS)

Jung, Sung Suk; Sohn, Il

2012-12-01

The crystallization behavior of a calcium-aluminate system with various MgO content from 2.5 to 7.5 wt pct and CaO/Al2O3 ratios between 0.8 and 1.2 has been examined using a confocal laser scanning microscope (CLSM). CCT (continuous cooling transformation) and time temperature transformation (TTT) diagrams were constructed to identify the primary crystal phase of slag at different compositions and at cooling rates between 25 and 800 K/minutes. In the slag at a CaO/Al2O3 ratio of 1.0, crystallization temperature increased during isothermal and continuous cooling with higher MgO content, and the shortest incubation time was observed at 5 wt pct MgO. When MgO content was fixed to be 5 wt pct, crystallization temperature increased with lower CaO/Al2O3 ratio. According to the slag composition, cooling rates and temperature, the primary phase could be CA, or C5A3, or C3A, or C3MA2, or MgO, and the crystal morphology changes from dendrites to faceted crystals to columnar crystals in this composition range.

Evaluation of transdermal delivery of nanoemulsions in ex vivo porcine skin using two-photon microscopy and confocal laser-scanning microscopy

NASA Astrophysics Data System (ADS)

Choi, Sanghoon; Kim, Jin Woong; Lee, Yong Joong; Delmas, Thomas; Kim, Changhwan; Park, Soyeun; Lee, Ho

2014-10-01

This study experimentally evaluates the self-targeting ability of asiaticoside-loaded nanoemulsions compared with nontargeted nanoemulsions in ex vivo experiments with porcine skin samples. Homebuilt two-photon and confocal laser-scanning microscopes were employed to noninvasively examine the transdermal delivery of two distinct nanoemulsions. Prior to the application of nanoemulsions, we noninvasively observed the morphology of porcine skin using two-photon microscopy. We have successfully visualized the distributions of the targeted and nontargeted nanoemulsions absorbed into the porcine skin samples. Asiaticoside-loaded nanoemulsions showed an improved ex vivo transdermal delivery through the stratum corneum compared with nonloaded nanoemulsions. As a secondary measure, nanoemulsions-applied samples were sliced in the depth direction with a surgical knife in order to obtain the complete depth-direction distribution profile of Nile red fluorescence. XZ images demonstrated that asiaticoside-loaded nanoemulsion penetrated deeper into the skin compared with nontargeted nanoemulsions. The basal layer boundary is clearly visible in the case of the asiaticoside-loaded skin sample. These results reaffirm the feasibility of using self-targeting ligands to improve permeation through the skin barrier for cosmetics and topical drug applications.

Forest Resource Measurements by Combination of Terrestrial Laser Scanning and Drone Use

NASA Astrophysics Data System (ADS)

Cheung, K.; Katoh, M.; Horisawa, M.

2017-10-01

Using terrestrial laser scanning (TLS), forest attributes such as diameter at breast height (DBH) and tree location can be measured accurately. However, due to low penetration of laser pulses to tree tops, tree height measurements are typically underestimated. In this study, data acquired by TLS and drones were combined; DBH and tree locations were determined by TLS, and tree heights were measured by drone use. The average tree height error and root mean square error (RMSE) of tree height were 0.8 and 1.2 m, respectively, for the combined method, and -0.4 and 1.7 m using TLS alone. The tree height difference was compared using airborne laser scanning (ALS). Furthermore, a method to acquire 100 % tree detection rate based on TLS data is suggested in this study.

A large capacity time division multiplexed (TDM) laser beam combining technique enabled by nanosecond speed KTN deflector

NASA Astrophysics Data System (ADS)

Yin, Stuart (Shizhuo); Chao, Ju-Hung; Zhu, Wenbin; Chen, Chang-Jiang; Campbell, Adrian; Henry, Michael; Dubinskiy, Mark; Hoffman, Robert C.

2017-08-01

In this paper, we present a novel large capacity (a 1000+ channel) time division multiplexing (TDM) laser beam combining technique by harnessing a state-of-the-art nanosecond speed potassium tantalate niobate (KTN) electro-optic (EO) beam deflector as the time division multiplexer. The major advantages of TDM approach are: (1) large multiplexing capability (over 1000 channels), (2) high spatial beam quality (the combined beam has the same spatial profile as the individual beam), (3) high spectral beam quality (the combined beam has the same spectral width as the individual beam, and (4) insensitive to the phase fluctuation of individual laser because of the nature of the incoherent beam combining. The quantitative analyses show that it is possible to achieve over one hundred kW average power, single aperture, single transverse mode solid state and/or fiber laser by pursuing this innovative beam combining method, which represents a major technical advance in the field of high energy lasers. Such kind of 100+ kW average power diffraction limited beam quality lasers can play an important role in a variety of applications such as laser directed energy weapons (DEW) and large-capacity high-speed laser manufacturing, including cutting, welding, and printing.

Detection of lead in brass by laser-induced breakdown spectroscopy combined with laser-induced fluorescence

NASA Astrophysics Data System (ADS)

Goueguel, Christian; Laville, Stéphane; Loudyi, Hakim; Chaker, Mohamed; Sabsabi, Mohamad; Vidal, François

2008-06-01

Laser-Induced Breakdown Spectroscopy (LIBS) technique combined with Laser-Induced Fluorescence (LIF) is known to be a high sensitivity and high selectivity analytical technique. Although sub-ppm limits of detection (LoD) have already been demonstrated, there is still a constant and urgent need to reach lower LoDs. Here, we report results obtained for the detection of lead trace in brass samples. The plasma was produced by a Q-switched Nd:YAG laser at 1064 nm and then re-excited by a nanosecond optical parametric oscillator (OPO) laser tuned at 283.31 nm. Emission from Pb atoms was then observed at 405.78 nm. The experiments were performed in air at atmospheric pressure. We found out that the optimal conditions were obtained for an ablation fluence of 2-3 J/cm2 and inter-pulse delay of 8-10 μs. Also, excitation energy of about 200 μJ was required to maximize the Pb(I) 405.78 nm emission. Using the LIBS-LIFS technique, the LoD was estimated to be about 180 ppb over 100 laser shots, which corresponds to an improvement of about two orders of magnitude with that obtained using conventional LIBS.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

PubMed

Chakraborty, Nilay; Wang, Mian; Solocinski, Jason; Kim, Wonsuk; Argento, Alan

2016-01-01

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera's collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

Confocal microscopy using variable-focal-length microlenses and an optical fiber bundle.

PubMed

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

2005-10-01

The use of variable-focal-length (VFL) microlenses can provide a way to axially scan the foci across a sample by electronic control. We demonstrate an approach to coupling VFL microlenses individually to a fiber bundle as a way to create a high-throughput aperture array with a controllable aperture pattern. It would potentially be applied in real-time confocal imaging in vivo for biological specimens. The VFL microlenses that we used consist of a liquid-crystal film sandwiched between a pair of conductive substrates for which one has a hole-patterned electrode. One obtains the variation of the focal length by changing the applied voltage. The fiber bundle has been characterized by coupling with both coherent and incoherent light sources. We further demonstrate the use of a VFL microlens array in combination with the fiber bundle to build up a confocal system. The axial response of the confocal system has been measured without mechanical movement of the sample or the objective, and the FWHM is estimated to be approximately 16 microm, with asymmetric sidelobes.

Closure of skin incisions by laser-welding with a combination of two near-infrared diode lasers: preliminary study for determination of optimal parameters

NASA Astrophysics Data System (ADS)

Hu, Liming; Lu, Zhihua; Wang, Biao; Cao, Junsheng; Ma, Xiaobo; Tian, Zhenhua; Gao, Zhijian; Qin, Li; Wu, Xiaodong; Liu, Yun; Wang, Lijun

2011-03-01

Laser welding has the potential to become an effective method for wound closure and healing without sutures. Closure of skin incisions by laser welding with a combination of two near-infrared lasers (980 and 1064 nm), was performed for the first time in this study. One centimeter long, full-thickness incisions were made on the Wistar rat's dorsal skin. The efficiencies of laser-welding with different parameters were investigated. Incision-healing, histology examination, and a tensile strength test of incisions were recorded. Laser welding with the irradiance level of 15.9 W/cm2 for both 980 and 1064-nm lasers and exposure time of 5 s per spot in continuous wave mode yielded a more effective closure and healing with minimal thermal damage, faster recovery, and stronger apposition in comparison with a suturing technique. The conclusion is that skin welding with a combination of two near-infrared diode lasers can be a good candidate for incision closure, and further investigations are in progress for clinical use.

Closure of skin incisions by laser-welding with a combination of two near-infrared diode lasers: preliminary study for determination of optimal parameters.

PubMed

Hu, Liming; Lu, Zhihua; Wang, Biao; Cao, Junsheng; Ma, Xiaobo; Tian, Zhenhua; Gao, Zhijian; Qin, Li; Wu, Xiaodong; Liu, Yun; Wang, Lijun

2011-03-01