Fiberoptic Illumination Research Articles

An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes

IntroductionHuman induced pluripotent stem cell-derived cardiomyocytes are available from various sources and they are being evaluated for safety testing. Several platforms are available offering different assay principles and read-out parameters: patch-clamp and field potential recording, imaging or photometry, impedance measurement, and recording of contractile force. Routine use will establish which assay principle and which parameters best serve the intended purpose. MethodsWe introduce a combination of field potential recording and calcium ratiometry from spontaneously beating cardiomyocytes as a novel assay providing a complementary read-out parameter set. Field potential recording is performed using a commercial multi-well multi-electrode array platform. Calcium ratiometry is performed using a fiber optic illumination and silicon avalanche photodetectors. Data condensation and statistical analysis are designed to enable statistical inference of differences and equivalence with regard to a solvent control. ResultsSimultaneous recording of field potentials and calcium transients from spontaneously beating monolayers was done in a nine-well format. Calcium channel blockers (e.g. nifedipine) and a blocker of calcium store release (ryanodine) can be recognized and discriminated based on the calcium transient signal. An agonist of L-type calcium channels, FPL 64176, increased and prolonged the calcium transient, whereas BAY K 8644, another L-type calcium channel agonist, had no effect. Both FPL 64176 and various calcium channel antagonists have chronotropic effects, which can be discriminated from typical “chronotropic” compounds, like (±)isoprenaline (positive) and arecaidine propargyl ester (negative), based on their effects on the calcium transient. DiscussionDespite technical limitations in temporal resolution and exact matching of composite calcium transient with the field potential of a subset of cells, the combined recording platform enables a refined interpretation of the field potential recording and a more reliable identification of drug effects on calcium handling.

Fiber optic illumination of a poly(dimethylsiloxane) sheath flow cuvette for diode laser induced fluorescence detection in capillary electrophoresis

A Tee configuration sheath flow cuvette with square cross-section channels has been produced in PDMS for CE detection. The output of a 1.4 W laser diode operating at 450 nm was focused onto the 300 μm core of a 370 μm od fiber optic whose end was inserted into one arm of the Tee for LIF. The optimal configuration had the fiber optic positioned 500 μm downstream from the intersection and the end of the 35 cm 50 μm id 365 μm od capillary just outside the intersection and in the leg of the Tee, resulting in a 90° configuration. Detection limits of 50 and 3 pM and linear calibrations of at least three orders of magnitude were obtained for Lucifer Yellow and fluorescein, respectively.

Performance of Series Connected GaAs Photovoltaic Converters under Multimode Optical Fiber Illumination

In many military and industrial applications, GaAs photovoltaic (PV) converters are connected in series in order to generate the required voltage compatible with most common electronics. Multimode optical fibers are usually used to carry high-intensity laser and illuminate the series connected GaAs PV converters in real time. However, multimode optical fiber illumination has a speckled intensity pattern. The series connected PV array is extremely sensitive to nonuniform illumination; its performance is limited severely by the converter that is illuminated the least. This paper quantifies the effects of multimode optical fiber illumination on the performance of series connected GaAs PV converters, analyzes the loss mechanisms due to speckles, and discusses the maximum illumination efficiency. In order to describe the illumination dependent behavior detailedly, modeling of the series connected PV array is accomplished based on the equivalent circuit for PV cells. Finally, a series of experiments are carried out to demonstrate the theory analysis.

Removal of indoor α-pinene with a fiber optic illuminated honeycomb monolith photocatalytic reactor

This study was undertaken to investigate the influencing factors including gas flow rate, inlet α-pinene concentration and relative humidity on the removal of α-pinene in a Degussa P25 supported honeycomb monolith reactor. We used the fiber optic illumination to enhance the intensity of UV-light irradiating on the Degussa P25 photocatalyst. The α-pinene conversion increased with the increase of gas flow rate indicating that the reaction rate was associated with the gaseous phase mass transfer. The α-pinene conversion varied between 91% and 96% in the range of inlet α-pinene concentration (400–2400 ppb) and relative humidity (30–70%) examined. The kinetics fits the Langmuir–Hinshelwood model. The rate coefficient (k) of α-pinene under RH30%, 50% and 70% was 0.82, 0.24, and 0.18 μmol m−2s−1, respectively. The competitive Langmuir adsorption constants for α-pinene under RH30%, 50% and 70% were 0.17, 0.56 and 1.74 ppm−1, respectively. The effect of relative humidity on α-pinene conversion depends on the inlet α-pinene concentration and raising relative humidity in sum has a positive effect on the reduction of partially oxidized intermediates within the range investigated.

Enhancement of the Spatial Resolution of the Chemical Imaging Sensor by a Hybrid Fiber-Optic Illumination

The chemical imaging sensor, which is based on the principle of the light-addressable potentiometric sensor (LAPS), is a powerful tool to visualize the spatial distribution of chemical species on the sensor surface. The spatial resolution of this sensor depends on the diffusion of photocarriers excited by a modulated light. In this study, a novel hybrid fiber-optic illumination was developed to enhance the spatial resolution. It consists of a modulated light probe to generate a photocurrent signal and a ring of constant light, which suppresses the lateral diffusion of minority carriers excited by the modulated light. It is demonstrated that the spatial resolution was improved from 92μm to 68μm.

Giants of orthopaedic surgery: Masaki Watanabe MD.

“The real voyage of discovery consists not in seeking new landscapes, but in having new eyes,” Marcel Proust said. On May 4, 1962, Masaki Watanabe MD opened new eyes. On that day, he performed the first arthroscopic menisectomy using the arthroscopic instruments he developed [7, 14, 22]. The patient was a 17-year-old boy who twisted his knee playing basketball. Watanabe debrided the flap tear of the medial meniscus. The patient went home the same day. In 6 weeks, he was back playing basketball [22]. A pioneer in the development of the modern arthroscope with fiberoptic illumination, Watanabe is widely considered the “father of arthroscopy.” (Fig. 1) [8] His arthroscope was a specialized endoscope, derived from a rudimentary cystoscope. The humble orthopaedic surgeon from Japan rarely spoke of his own personal achievements, so his peers did the talking for him, championing his instruments and techniques at international orthopaedic meetings. Fig. 1 Watanabe (left) with his former student Prof. Hideshige Moriya (right) in June 1980. Watanabe is widely considered the “father of arthroscopy” Arthroscopic surgery is now commonplace—an indispensible outpatient procedure in many parts of the world. The benefits of minimally invasive surgery and early mobilization afforded by arthroscopic surgery are accepted as the standard of care. It was a long road to commonplace. The development of the arthroscope is a story of mentorship, dedication, and passion. Watanabe, among many others, revolutionized orthopaedic surgery. The quest to look into body cavities with small incisions dates to Pompeii, but the modern version of the arthroscope emerged from Japan.

Technology development of the soft X-ray tomography system in Wendelstein 7-X stellarator

The engineering and design of the soft X-ray Multi Camera Tomography System (XMCTS) in Wendelstein 7-X stellarator (W7-X) must fulfill several additional requirements compared to short pulse machines. The XMCTS has to withstand irradiation and electron cyclotron microwave loads in addition to being ultra high vacuum compatible, having low magnetic permeability and using low neutron activation materials (e.g. Co≤2000ppm). A further difficulty is the limited space inside the plasma vessel, which requires special engineering solutions.After detailed design development, supported by finite element analyses, prototypes have been manufactured and tested. At the end all test results have successfully proven that the components fulfill the requirements and that reliable and stable measurements will be possible with the XMCTS diagnostics during W7-X operation.The paper describes the design and the technological development, in particular on the electric multipin feedthrough (UHV barrier between in vessel detectors and the preamplifiers), the active cooling of the electronic components (reducing dark current/noise increase), the pneumatic shutter (protection of the detectors from sputtering and during baking) and the fiber optics illumination system (calibration of the detectors).

ライトガイドおよび涙道内視鏡下に施行する副鼻腔粘液嚢胞手術

It is hard to cure dacryocystitis caused by a paranasal sinus mucocele with treatment which only targets the mucocele. Also, it is difficult to identify the lacrimal sac and the nasolacrimal duct preoperatively and intraoperatively when the lacrimal passage is markedly changed by the mucocele or previous surgery. We experienced four cases of mucocele complicated by lacrimal stenosis or obstruction. We performed marsupialization of the mucocele and direct silicon intubation or endoscopic dacryocystorhinostomy simultaneously with the use of a fiberoptic illuminator or dacryoendoscopy. Assisted by those devices, lacrimal procedures can now be done quickly and safely regardless of the surgeon's experience. In addition, performing surgeries both for the lacrimal passage and for the mucocele at the same time can minimize the burden on patients.

Visibility enhancement of common bile duct for laparoscopic cholecystectomy by vivid fiber-optic indication: a porcine experiment trial

Bile duct injury (BDI) is the most serious iatrogenic complication during laparoscopic cholecystectomy (LC) and occurs easily in inexperienced surgeons since the position of common bile duct (CBD) and its related ductal junctions are hard to precisely identify in the hepatic anatomy during surgery. BDI can be devastating, leading to chronic morbidity, high mortality, and prolonged hospitalization. In addition, it is the most frequent injury resulting in litigation and the most likely injury associated with a successful medical malpractice claim against surgeons. This study introduces a novel method for conveniently and rapidly indicating the anatomical location of CBD during LC by the direct fiber-optic illumination of 532-nm diode-pumped solid state laser through a microstructured plastic optical fiber to avoid the wrong identification of CBD and the injury from mistakenly cutting the CBD that can lead to permanent and even life threatening consequences. Six porcine were used for preliminary intra-CBD illumination experiments via laparotomy and direct duodenal incision to insert the invented CBD illumination laser catheter with nonharmful but satisfactory visual optical density.

A feasibility study on unsaturated flow bioreactor using optical fiber illumination for photo-hydrogen production

An unsaturated flow bioreactor with Rhodopseudomonas palustris CQK 01 was developed for photo-hydrogen production. The bioreactor developed in this work consisted of a reaction bed packed with spherical glass beads and internal optical fiber that provided uniform light distribution and achieved high light transmission efficiency. Unsaturated conditions were obtained by filling both argon and nutrient solution into the bioreactor. Experimental results demonstrated the feasibility of the biofilm formation in an unsaturated environment. With the formed biofilm, parametric studies on the photo-hydrogen production performance under different liquid flow rates and initial substrate concentrations were conducted. It was found that the performance of the bioreactor came to the optimal state at the liquid flow rate of 500 mL/h. Moreover, such a design enabled the bioreactor to be operated at relatively high glucose concentration without the substrate inhibition, yielding a good biohydrogen production performance.

On-line and real time cell counting and viability determination for animal cell process monitoring by in situ microscopy

BackgroundTwo of the key parameters to be monitored during cellcultivation processes are cell concentration and viability.Until today, this is very often done off-line by sterilesampling and subsequent counting using a hemocyt-ometer or an electronic cell counter. Cell biology lacks ameasurable quantity by which single cells in suspensioncan be non-invasively diagnosed as dead or alive. How-ever, it would be of significance for process monitoringand in the light of initiatives like PAT if cell density aswell as viability could be determined directly and on-line.Optical measurement of cell density byin situ micro-scopy eliminates the need for sampling and allows forcontinuous monitoring of this key parameter; see e.g.[1,2]; Guez et al. [1] describe an in situ microscope(ISM) which does not use any moving mechanical partswithin or outside the fermentation vessel. It transmits inreal time images taken directly in the stirred suspensionwithin the bioreactor. Image data is processed and eval-uated to provide monitoring of cell-density and mor-phological parameters, e.g. cell size, by means ofassessing the obtained in situ cell-micrographs.Previously, we have extendedin situ microscopytowards viability assessment of suspended cells [3,5].Now, we present new findings on this topic and showthat in cultures of suspended cells, cell-death corre-sponds to measurable changes in morphometric para-meters as e.g. variance, contrast or entropy of thegreyvalues of in situ cell-micrographs. As an example,here we show viability determination via greyvaluedispersion.Material and methodsWe use a custom built high resolution ISM (HS Man-nheim) with water immersion objective, 40x magnifica-tion, numerical aperture 0.75 equipped with optical fiberillumination. Data acquisition is at 0.3– 15 frames persecond, frames have 1293x1040 pixels; primary dataanalysis results in cell micrographs (Figure 1a). We haveapplied the system to bench top and larger bioreactors(see e.g. [4]) and worked mainly with Jurkat, CHO andHybridoma cells.For the experiment presented in Figure 1a/b, cells arecultivated in a Biostat C30 (Sartorius BBI Systems) withthe ISM inserted in one of the existing probe ports. Pro-prietary hybridoma cells (InVivo BioTech Services) arecultured in serum free ISF-1 (InVivo BioTech Services;Biochrom AG) and monitored over the full length ofthe fermentation (not shown).For the experiment presented in Figure 1c, cells arecultivated in a custom built autoclavable steel bench topreactor(HSMannheim)with25mmporttoaccommo-date the ISM and a working volume of 0.7 L. To testreal time and viability determination capabilities of thesystem over a wide range of viabilities in a short time,cells were challenged with 3% Ethanol at 42 hours. Cellcounts (not shown) and viability were determined by insitu microscopy and, as reference, by means of a ViCellcell viability analyser (Beckman Coulter) and Flow Cyto-metry (Partec) using Annexin V / FITC and PI staining.Jurkat cells (DSMZ ACC 282) were cultured in 90%RPMI 1640 + 10% FBS.

The effect of a gap-junction blocker, carbenoxolone, on ischemic brain injury and cortical spreading depression

Cortical spreading depression (CSD) has been shown to cause secondary cell loss in experimental models of brain injury and in patients, and blocking of CSD is a potential neuroprotective strategy. Here we tested the hypothesis that gap junctions affect CSD under physiological conditions as well as infarct development in a rat two-vein occlusion model suited to study pathophysiology of the penumbra (n = 71). We applied the gap junction blocker carbenoxolone (CBX) or saline intra-ventricularly. Interestingly, CBX temporarily increased systemic blood pressure and cortical blood flow (41% and 53%, 15 min after 250 μg CBX). We induced CSD with cortical microinjection of potassium chloride (KCl), counted how many spontaneous CSDs after CSD induction were elicited and measured the propagation velocity. After 250 μg CBX administration, significant 37.5 ± 6.5 additional CSDs were seen. CSD velocity increased significantly after 50 μg and 250 μg CBX. Occlusion of two adjacent cortical veins using Rose Bengal dye and fiberoptic illumination followed by 250 μg CBX or saline showed a significant more than doubling of infarct volumes 7 days after CBX. The current experiments provide evidence that CBX can accelerate the initiation and propagation of CSD suggesting opening of gap junctions is not required for CSD propagation. Blocking gap junctions worsens outcome from focal cerebral ischemia. Hence, measures intended to improve spatial buffering via astroglial gap junctions could have therapeutic potential in disease processes involving CSD.

Single Molecule Studies of Nuclear Transport

The translocation of single biomolecules through nanopores is one of the most fundamental processes of life. For example, the import of histones into the cell nucleus via the nuclear pore complex (NPC) is crucial for DNA condensation (1,2). We aim to gain a deeper understanding of NPC translocation on a single molecule level by combining optical tweezers with patch clamp techniques. This gives us the unique possibility to analyze the interaction forces between a biological macromolecule and the NPC while measuring the relevant time scales by electrophysiological recordings. We present preliminary results on the characterisation of the properties of NPCs concerning mode and duration of macromolecular translocation. Nuclei extracted from the syntactical coconut cocos nucifera are one model system. We demonstrate the selectivity of the NPC using an import signal tagged with GFP. We determine the conductance of a single nuclear pore to be approximately 490pS. Our results allow for estimation of the nuclear pore density to be between one and four per μm2. From observations of the ionic current across the pores, we are able to observe gating and to derive the translocation time of cargo across single NPCs . As a technical development towards nuclear pore force spectroscopy we introduce optical fiber illumination for real-time tracking of optically trapped particles. We demonstrate video-based fluctuation analysis of a single colloid at 10,000 Hz in order to study the dynamics of an attached biological macromolecule with microsecond time resolution (3-5).

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.

Real-time particle tracking at 10,000 fps using optical fiber illumination

We introduce optical fiber illumination for real-time tracking of optically trapped micrometer-sized particles with microsecond time resolution. Our light source is a high-radiance mercury arc lamp and a 600 μm optical fiber for short-distance illumination of the sample cell. Particle tracking is carried out with a software implemented cross-correlation algorithm following image acquisition from a CMOS camera. Our image data reveals that fiber illumination results in a signal-to-noise ratio usually one order of magnitude higher compared to standard Köhler illumination. We demonstrate position determination of a single optically trapped colloid with up to 10,000 frames per second over hours. We calibrate our optical tweezers and compare the results with quadrant photo diode measurements. Finally, we determine the positional accuracy of our setup to 2 nm by calculating the Allan variance. Our results show that neither illumination nor software algorithms limit the speed of real-time particle tracking with CMOS technology.

An improved multiple-site optical membrane potential-recording system to obtain high-quality single sweep signals in intact rat cerebral cortex

We improved our optical recording system to record epifluorescence optical signals from the intact cerebral cortex. Using custom-made fiber optic illumination equipment and tandem lens optics excluding a dichroic mirror, we successfully obtained high-quality single sweep optical signals in vivo in rat sensorimotor cortex. After reduction of pulsation artifacts with software originally designed for prolonged continuous recording under spontaneous breathing, the signal-to-noise ratio of the optical signal was sufficient to analyse evoked responses quantitatively. We were able to make isochrones for the onset of responses based on single sweep optical recordings. We further examined the effect of baclofen using this isochrone map, and succeeded in quantitatively demonstrating the inhibitory effect on the spatial pattern of neural activity in the rat sensorimotor cortex by calculating the area of response region on the isochrone map. Thus, our improved system provides sustained single sweep records suitable for quantitative analysis using epifluorescence optics.

Prevention of Biliary Duct Injury in Laparoscopic Cholecystectomy Using Optical Fiber Illumination in Common Bile Duct.

BackgroundBiliary duct injury (BDI) is one of the most common complications in laparoscopic cholectecystomy (LC), in this study, we have tried to place an illuminating optical fiber via endoscopy in the CBD during LC, the biliary duct anatomy can be clearly delineated, thus CBD injury is avoided.MethodsSixteen patients with chronic cholecystitis or/and cholelithiasis from February 2007 to June 2008 were performed LC with placement of optical fiber in CBD, the fiber with cold light illuminates the whole extrahepatic biliary system. Three 6-mm titanium clips were applied to the soft tissue surrounding the hepatic duct, CBD and the cystic duct confluence with CBD, respectively; one titanium clip was applied to the surface of cystic duct near the infundibulum of gallbladder. The cytic duct, CBD and common hepatic duct were clearly identified and delineated in the operating field and LC was performed.ResultsAll the 16 patients were performed LC using this procedure successfully, there were no LC-related complications, nor complications related to endoscopic retrograde cholangiopanceatography (ERCP).ConclusionsThe endoscopically placed optical fiber in the CBD can clearly identify the CBD, Calot’s triangle and the common hepatic duct, this can reduce the bile duct injury in LC and imporve the safety of LC.

Blue-green spectral minimum correlates with oxyhemoglobin saturation in vivo

An imaging multi-spectral retinal oximeter with intravitrial illumination is used to perform the first in vivo test of the blue-green minima shift oximetry method (BGO) in swine eyes [K. R. Dennighoff, R. A. Chipman, and L. W. Hillman, Opt. Lett. 31, 924-926 (2006); J. Biomed. Opt. 12, 034020 (2007).] A fiber optic intravitreal illuminator inserted through the pars plana was coupled to a monochromator and used to illuminate the retina from an angle. A camera viewing through the cornea recorded a series of images at each wavelength. This intravitreal light source moves the specular vessel glint away from the center of the vessel and directly illuminates the fundus behind most blood vessels. These two conditions combine to provide accurate measurements of vessel and perivascular reflectance. Equations describing these different light paths are solved, and BGO is used to evaluate large retinal vessels. In order to test BGO calibration in vivo, data were acquired from swine with varied retinal arterial oxyhemoglobin saturations (60-100% saturation.). The arterial saturations determined using BGO to analyze the multispectral image sets showed excellent correlation with co-oximeter data (r2=0.98, and residual error +/-3.4% saturation) and are similar to results when hemoglobin and blood were analyzed using this technique.

Diffuse spectral fundus reflectance measured using subretinally placed spectralon

The diffuse fundus reflectance and the spectral transmittance of the swine sensory retina was measured in vivo using intravitreal illumination. Pars plana vitrectomy and intravitreal manipulations were performed on a female American Yorkshire domestic swine. Light from a scanning monochromator was coupled into a fiber optic intraocular illuminator inserted into the vitreous. A 1.93-mm(2) region of the illuminated fundus was imaged from an oblique illumination angle. Multispectral retinal images were acquired for four experimental conditions: the eye (1) prior to vitrectomy, (2) after vitrectomy, (3) after insertion of a Spectralon disk super-retinally, and (4) after subretinal insertion of the disk. The absorption of melanin and hemoglobin in the red wavelengths was used to convert relative spectral reflectance to absolute reflectance. The flux scattered from the super-retinal Spectralon was used to correct for scattering in the globe. The transmittance of the sensory retina was measured in vivo using the scatter corrected subretinal Spectralon disk reflectance. The hemoglobin and melanin components of the spectrum due to scattered light were removed from the retinal transmission spectrum. The in vivo spectral transmittance of the sensory retina in this swine was essentially flat across the visible spectrum, with an average transmittance >90%.

An LED-based illuminator for microphotometric studies of cells

A fiber-optic illuminator with superbright semiconductor LEDs for single-cell microphotometry is described. The illuminator provides excitation at wavelengths of 385, 440, 480, and 530 nm (30, 70, 50, and 20 mW, respectively). The long-term instability of its radiant power is ≤0.1 %/h, and its service life is 30000 h. The characteristics of the LED-based illuminator are compared to those of a commercial illuminator based on a DeltaRAM scanning monochromator (Photon Technology International, United States).