Biomedical subjects

I Klimant

Publications and source records attributed to I Klimant.

12 recordsLinked to original sources

PubMed2006

Filter cubes with built-in ultrabright light-emitting diodes as exchangeable excitation light sources in fluorescence microscopy.

The use of ultrabright light-emitting diodes as a potential substitute for conventional excitation light sources in fluorescence microscopy is demonstrated. We integrated ultrabright light-emitting diodes in the filter block of a conventional fluorescence microscope together with a collimating Fresnel lens, a holographic diffuser and emission filters. This setup enabled convenient changes between different excitation light sources and resulted in high excitation efficiencies. Quantitative comparison of image intensities of test samples revealed that light-emitting diodes yielded intensities in the range of a mercury arc lamp depending on the wavelength. The use of ultrabright light-emitting diodes also enabled luminescence lifetime imaging without the need for image intensification.

Journal Article↗

PubMed2001

A new type of phosphorescent nanospheres for use in advanced time-resolved multiplexed bioassays.

A new concept to design phosphorescent nanospheres is presented. The spheres are distinguishable by their individual decay time and spectral distribution of their emission spectra. They are composed of a phosphorescent ruthenium metal-ligand complex (MLC) dissolved, along with certain strongly fluorescent cyanine dyes, in modified polyacrylonitrile-based nanospheres. Since the emission spectrum of the MLC overlaps the absorption spectrum of the cyanine and both the MLC (the donor) and the cyanine (the acceptor) are in close spatial proximity, efficient resonance energy transfer (RET) does occur. Thus, the nanospheres emit dual luminescence, one from the acceptor dye and the other from the donor MLC. Variation of the concentrations of the acceptor dye results in a varying efficiency of RET, thus making the spheres distinguishable. Hence, a set of multiplexable sphere labels is obtained by using one MLC (acting as the phosphorescent donor and present in constant concentration) and one acceptor dye (which varies in terms of both spectral properties and concentration). The nanospheres can be identified by the emission maximum (reflecting the kind of acceptor dye) and by decay time (reflecting its concentration). Since the same donor MLC is used throughout, all nanospheres can be excited with the same light source.

Biological Assay↗

PubMed2001

Fluorescent imaging of pH with optical sensors using time domain dual lifetime referencing.

We present a referenced scheme for fluorescence intensity measurements that is useful for imaging applications. It is based on the conversion of the fluorescence intensity information into a time-dependent parameter. A phosphorescent dye is added in the form of approximately 10-microm particles to the sample containing the pH-sensitive fluorescent indicator. Both the reference dye and the pH probe are excited simultaneously by a blue LED, and an overall luminescence is measured. In the time-resolved imaging method presented here, two images taken at different time gates were recorded using a CCD camera. The first image is recorded during excitation and reflects the luminescence signal of both the fluorophore (pH) and the phosphor (reference). The second image, which is measured after a certain delay (after switching off the light source), is solely caused by the long-lived phosphorescent dye. Because the intensity of the fluorophore contains the information on pH, whereas phosphorescence is pH-independent, the ratio of the images displays a referenced intensity distribution that reflects the pH at each picture element (pixel). The scheme is useful for LED light sources and CCD cameras that can be gated with square pulses in the microsecond range. The fundamentals and potential of this new method, to which we refer as time domain dual lifetime referencing (t-DLR), are demonstrated.

Journal Article↗

PubMed2001

Dual lifetime referencing as applied to a chloride optical sensor.

A membrane with an optical response to chloride has been developed that contains two luminophores that display two largely different decay times. The first luminophore (the "reference") is a chloride-insensitive ruthenium metal-ligand complex possessing a decay time in the microsecond range. The second luminophore is the short-lived chloride-quenchable fluorescent probe lucigenin. Both are contained in a hydrogel matrix and are excited by a blue LED emitting sinusoidally modulated light. Under these conditions, the chloride-dependent fluorescence intensity of lucigenin can be converted in an analyte-dependent fluorescence phase shift that depends on the ratio of the two luminescence intensities and can be measured at modulation frequencies of typically 45 kHz. The dynamic range of this sensor can be adjusted by either varying the ratio of the two luminophores or selecting a particular optical filter combination.

Acridines↗

PubMed2001

Fluoro reactants and dual luminophore referencing: a technique to optically measure amines.

An optical sensor for aqueous 1-butylamine is presented which combines two novel techniques: A fluorescent indicator dye (fluoro reactand) embedded in a thin polymer layer performs a reversible chemical reaction with the analyte, causing changes in luminescence intensity. At the same time, inert phosphorescent beads dispersed within the polymer layer provide luminescence signals that act as an internal reference for the indicator dye. As a consequence, the optical sensor is independent of light source fluctuations, ambient light, drifts in optoelectronic setup, or optical fiber bending.

Journal Article↗

PubMed2000

Fiber-optic microsensor for high resolution pCO2 sensing in marine environment.

A fast responding fiber-optic microsensor for sensing pCO2 in marine sediments with high spatial resolution is presented. The tip diameter varies typically between 20 and 50 microm. In order to make the pH-indicator 8-hydroxypyrene-1,3,6-trisulfonate soluble in the ethyl cellulose matrix, it was lipophilized with tetraoctylammonium as the counterion [HPTS-(TOA)4]. The microsensor was tuned to sense very low levels of dissolved carbon dioxide which are typically present in marine systems. The detection limit is 0.04 hPa pCO2 which corresponds to 60 ppb CO2 of dissolved carbon dioxide. A soluble Teflon derivative with an extraordinarily high gas permeability was chosen as a protective coating to eliminate interferences by ionic species like chloride or pH. Response times of less than 1 min were observed. The performance of the new microsensor is described with respect to reproducibility of the calibration curves, dynamic range, temperature behavior, long term stability and storage stability. The effect of hydrogen sulfide as an interferent, which is frequently present in anaerobic sediment layers, was studied in detail.

Journal Article↗

PubMed2000

Sol-gel based glucose biosensors employing optical oxygen transducers, and a method for compensating for variable oxygen background.

Various types of thin-film glucose biosensors based on the use of the enzyme glucose oxidase (GOx) have been developed. The luminescent oxygen probe Ru(dpp)--whose emission is quenched by oxygen--is used to measure the consumption of oxygen. Three different combinations of oxygen transducer and sol-gel immobilized GOx were tested. In the first, GOx was sandwiched between a sol-gel layer doped with Ru(dpp) and a second sol-gel layer composed of pure sol-gel (the 'sandwich' configuration). In the second, a sol-gel layer doped with Ru(dpp) was covered with sol-gel entrapped GOx (the 'two-layer configuration'). In the third, both GOx and a sol-gel powder containing GOx were incorporated into a single sol-gel phase (the 'powder configuration'). In all cases, it was found to be essential to add sorbitol which results in a more porous sol-gel in which diffusion is not impaired. The sandwich configuration provides the highest enzyme activity and the largest dynamic range (0.1-15 mM), but suffers from a distinct decrease in sensitivity upon prolonged use. The two-layer configuration has the fastest response time (t90 = 50 s), while the 'powder configuration' provides the best operational lifetime. The storage stability of all configurations exceeds 4 months if stored at 4 degrees C. In an Appendix, equations are derived which describe the response of such sensors, how the effect of varying oxygen supply can be compensated for by making use of two sensors, one sensitive to oxygen only, the other to both oxygen and glucose, and how such sensors can be calibrated using two calibrators only.

Biosensing Techniques↗

PubMed1996

New instrumentation for optical measuring of oxygen in gas or dissolved in liquids.

The optical oxygen sensor is a novel device for the determination of oxygen in gases or dissolved in liquids. It is based on the measurement principle of fluorescence quenching, which is completely different from that of polarographic oxygen sensors (today the most widespread devices of oxygen detection). The new instrument offers features and advantages, which render it not only a realistic alternative, but, for specific applications, make it superior to existing electrochemical methods. The system is based on low-cost semiconductor devices (light-emitting diodes, photodiodes, low-cost analogue and digital components) and new LED-compatible oxygen-sensitive membranes. The flow cell of the instrument may be thermostatted and the sensor can be calibrated by a simple two-point calibration procedure. The optical oxygen sensor is particularly suitable for measuring dissolved oxygen in respirometry, since no oxygen is consumed by the device and the signal is independent of sample flowrate or stirring speed. Typical fields of application are monitoring of oxygen in ground and drinking water, in process control in bioreactors and in breath gas and blood gas analysis.

Biosensing Techniques↗

PubMed1996

A new in vivo fluorimetric technique to measure growth of adhering phototrophic microorganisms.

We developed a noninvasive rapid fluorimetric method for the investigation of growth of adhering (benthic) phototrophic microorganisms. The technique is based on the sensitive detection of the in vivo fluorescence of chlorophylls chlorophyll a and bacteriochlorophyll a and monitors increases in signal over time as an indicator for growth. The growth fluorimeter uses modulated excitation light of blue-light-emitting diodes and a photodiode as the detector. The light-emitting diodes are mounted geometrically in an aluminum housing for efficient and uniform illumination of the bottoms of the growth containers. The fluorimeter was characterized with respect to detection limit and dynamic range. This system is capable of resolving in vivo chlorophyll a concentrations of 0.5 (mu)g liter(sup-1) in cyanobacteria and 0.03 (mu)g liter(sup-1) in diatoms as well as in vivo bacteriochlorophyll a concentrations in phototrophic bacteria of 0.3 (mu)g liter(sup-1), which points to an extremely high sensitivity compared with that of similar available techniques. Thus, the new fluorimeter allows the determination of growth at extremely low cell densities. The instrument was used successfully to measure the growth of several adhering isolates of the filamentous cyanobacterium Microcoleus chthonoplastes from benthic microbial mats in seawater of different salinities. The data obtained demonstrate broad growth responses for all strains, which thus can be characterized as euryhaline organisms.

Journal Article↗

PubMed1994

Optical triple sensor for measuring pH, oxygen and carbon dioxide.

A triple sensor unit consisting of opto-chemical sensors for measurement of pH, oxygen and carbon dioxide in bioreactors is presented. The pH and the CO2 sensor are based on the color change of a pH-sensitive dye immobilized on a polymeric support. The resulting changes in absorption are monitored through optical fibers. The oxygen sensor is based on the quenching of the fluorescence of a metal-organic dye. All three sensors are fully LED compatible. The sensitive membranes consist of plastic films and can be stored and replaced conveniently. The sensors are sterilizable with hydrogen peroxide and ethanol. In addition, the pH sensor is steam sterilizable. Accuracy, resolution and reproducibility fulfill the requirements for use in biotechnological applications. Calibration procedures for each sensor are presented. The working principle and the performance of all three sensors are described, with particular emphasis given to their application in bioreactors.

Carbon Dioxide↗

PubMedDate not supplied

Optical sensor for seawater salinity.

An optical sensor for the measurement of salinity in seawater has been developed. It is based on a chloride-quenchable fluorescent probe (lucigenin) immobilized on a Nafion film. Two approaches for measuring salinity via chloride concentration are presented. In the first, a change in salinity corresponds to a change in the fluorescence intensity of lucigenin. In the second, the fluorescence intensity information is converted into a phase angle information by adding an inert phosphorescent reference luminophore (a ruthenium complex entrapped in poly(acrylonitrile) beads). Under these conditions, the chloride-dependent fluorescence intensity of lucigenin can be converted into a chloride-dependent fluorescence phase shift which serves as the analytical information. This scheme is referred to as dual lifetime referencing (DLR). The sensor was used to determine the salinity in seawater and brackish water of the North Sea.

Journal Article↗

PubMedDate not supplied

Inert phosphorescent nanospheres as markers for optical assays.

A simple encapsulation technique is presented to produce highly phosphorescent, inert nanospheres that are suitable luminescent markers. It is based on the coprecipitation of phosphorescent ruthenium(II)-tris(polypyridyl) complexes and polyacrylonitrile (PAN) derivatives from a solution in N,N-dimethylformamide. The beads precipitate in the form of very small aggregates of spherical shape and a typical particle diameter of less than 50 nm. This process allows the encapsulation of phosphorescent and fluorescent dyes in an individual nanosphere provided that they are sufficiently lipophilic. Quenching by oxygen is negligible due to the use of PAN. The nanospheres were characterized with respect to their spectral properties (quantum yields of the luminophores, brightness, luminescence decay time), stability in aqueous buffered suspensions, and in terms of size, shape, and surface charge of the particles, as well as storage stability, quenching by oxygen, and dye leaching.

Acrylic Resins↗