Ingo Klimant

Optical measurement of oxygen and temperature in microscale: strategies and biological applications

Sediments, microbial mats, biofilms and other microbial communities are characterized by steep gradients of physical and chemical parameters. Microsensors are powerful tools to measure these parameters with a sufficient spatial resolution and with a small disturbance of the micro-environment in natural systems. Recently, fiber-optical microsensors have been introduced in the field of aquatic biology as an alternative to existing electrochemical microsensors. Such micro-optodes have already been developed for high-resolution measurement of dissolved oxygen and for temperature measurements. They are easy to fabricate and show an improved long-term and storage stability. An overview is given on the development and characterization of different types of micro-optodes for oxygen and temperature. A luminescence lifetime-based device has been developed which is portable and enables microsensing both in the laboratory and under field conditions. Limitations in practical work with optical microsensors are demonstrated, and strategies to overcome them briefly discussed. A micro-optode array as well as a method for high-resolution oxygen imaging in sediments are presented as two different ways to investigate the two-dimensional oxygen distribution in heterogeneous living systems. Future applications and developments in micro-optode research will be discussed briefly.

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.

A modular luminescence lifetime imaging system for mapping oxygen distribution in biological samples

We developed a new modular luminescence lifetime imaging system (MOLLI), that enables the imaging of luminescence lifetimes in the range of 1 ms to 1 s. The system can easily be adapted to different experimental applications. The central parts of the system are a recently released CCD-camera with a fast electronic shutter and gated LED (light emitting diode) or Xe excitation light sources. A personal computer controls the gating and image acquisition via a pulse delay generator. Here we present the new imaging system and give examples of its performance when used for measuring two-dimensional oxygen distributions with planar optodes. Furthermore, future applications of the system in biology are discussed.

Microsecond lifetime-based optical carbon dioxide sensor using luminescence resonance energy transfer

Abstract A lifetime-based optical sensor for the measurement of dissolved and gaseous carbon dioxide has been developed. The basic principle is radiationless energy transfer from ruthenium(II)-4,4′-diphenyl-2,2′-bipyridyl as luminescent donor to thymol blue (a common pH indicator) as acceptor, both embedded in a hydrophobic matrix. In the presence of carbon dioxide thymol blue is protonated and changes its color from blue to yellow resulting in a decrease in the rate of energy transfer and consequently an increase in decay time. The decay time as a p CO 2 -dependent parameter was measured in the frequency domain with a blue light emitting diode as a light source modulated at 75xa0kHz. The present sensor displays a phase shift up to 16° in the range 0–30xa0hPa of p CO 2 corresponding to a change in decay time from 0.38 to 1.05xa0μs. The detection limit was found to be 0.5xa0μM (22xa0ppb) of dissolved carbon dioxide and the response times are of the order of 15xa0s when going from 0 to 30xa0hPa p CO 2 . The temperature behavior of the sensor has been studied in detail and a linear relationship Arrhenius plot has been found. The effect of molecular oxygen as a potential quencher of the luminescence was investigated in detail. The carbon dioxide sensor is stable over weeks, and is robust.

A microoptode array for fine-scale measurement of oxygen distribution

Abstract A new microoptode array is presented that provides simultaneous measurement with eight oxygen microoptodes using a simple optical setup and a phase-angle detection principle. The measuring system consists of: (1) an optical unit with eight oxygen microoptodes, a special fiber-coupler array, optical filters, lenses, light sources (light-emitting diodes) and light detectors (photodiodes, photomultiplier tube); (2) a signal-processing unit with analog signal processing (phase-angle detection, filtering) and digital signal processing (control, data storage and display). The oxygen concentration is measured with tapered silica-glass fibers (tip diameter 20–30 μm) by the dynamic quenching of a luminophore. A phase-modulation technique is used to determine the phase-angle shift that is caused by the fluorescence lifetime when the indicator is excited sinusoidally. In a time multiplex mode each sensor signal is sampled. This multisensor array system is designed for the investigation of the oxygen distribution in biofilms and aquatic sediments. The new measuring system and first applications in artificial and natural systems are presented.

Measurement of chlorophyll fluorescence within leaves using a modified PAM Fluorometer with a fiber-optic microprobe

By using a fiber-optic microprobe in combination with a modified PAM Fluorometer, chlorophyll fluorescence yield was measured within leaves with spatial resolution of approximately 20 μm. The new system employs a miniature photomultiplier for detection of the pulse-modulated fluorescence signal received by the 20 μm fiber tip. The obtained signal/noise ratio qualifies for recordings of fluorescence induction kinetics (Kautsky effect), fluorescence quenching by the saturation pulse method and determination of quantum yield of energy conversion at Photosystem II at different sites within a leaf. Examples of the system performance and of practical applications are given. It is demonstrated that the fluorescence rise kinetics are distinctly faster when chloroplasts within the spongy mesophyll are illuminated as compared to palisade chloroplasts. Photoinhibition is shown to affect primarily the quantum yield of the palisade chloroplasts when excessive illumination is applied from the adaxial leaf side. The new system is envisaged to be used in combination with light measurements within leaves for an assessment of the specific contributions of different leaf regions to overall photosynthetic activity and for an integrative modelling of leaf photosynthesis.

Engineered Bacteria Based Biosensors for Monitoring Bioavailable Heavy Metals

This work presents an integrated analytical system based on immobilized engineered microorganisms and bioluminescence measurements for monitoring of bioavailable heavy metal ions (Cu being chosen as a model ion). A strain of microorganisms from Alcaligenes eutrophus (AE1239) was genetically engineered by inserting a luxCDABE operon from Vibrio fischeri under the control of a copper-induced promoter. As a result, copper ions induce bioluminescence, which is proportional to the concentration of the triggering ions, representing the basis of the design of the hereby described heavy metal biosensor. Microorganisms grown in two different media (Luria Broth and a modified mineral reconstitution medium/RM) were optimized and characterized in solution with regard to the influence of growth media and cell density in order to obtain optimal bioluminescent signals. Next, the microorganisms were immobilized in polymer matrices, compatible with fiber optics and were characterized with regard to sensitivity, selectivity, detection limit and storage stability. The lowest detection limit (1 µM) was achieved with microorganisms cultivated from glycerol stock solutions in the RM media and immobilized in a calcium alginate matrix.

Modeling of Mixing in 96-Well Microplates Observed with Fluorescence Indicators

Mixing in 96‐well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double‐disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 μL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition.

In Vivo Phosphorescence Imaging of pO2 Using Planar Oxygen Sensors

Objective: Oxygen‐dependent quenching of luminescence of metal porphyrin complexes has been used to image the pO2 distribution over tumor and normal tissue.

Recent progress in optical oxygen sensor instrumentation

Optical methods for the determination of dissolved or gaseous oxygen are mainly based on the principle of fluorescence quenching. Measurement schemes have been reported which employ various oxygen-sensitive dyes and bulky instrumentation. Typically, expensive fluorescence spectrometers or fibre-optic photometers have been used, and the applicability of such instruments is rather limited. A system based on low-cost semiconductor devices (light-emitting diodes (LEDS), photodiodes, low-cost analogue and digital components) and new LED-compatible oxygen-sensitive membranes has been developed at our institute. The instrument is capable of determining dissolved or gaseous oxygen and may be calibrated, for example, by a simple two-point calibration procedure with air-saturated and oxygen-free water. Thermostatization of the flow-through cell results in higher measurement accuracy and in a reduced influence of the ambient temperature on the instrument. The overall performance of the oxygen sensor has been investigated, e.g. measurement stability, effectivity of thermostatization, calibration, oxygen diffusion into the measuring cell and excitation feed-through.