Gerhard A. Holst

Luminescence Lifetime Imaging of Oxygen, pH, and Carbon Dioxide Distribution Using Optical Sensors

We present a modular system for time-resolved two-dimensional luminescence lifetime imaging of planar optical chemical sensors. It is based on a fast, gateable charge-coupled device (CCD) camera without image intensifier and a pulsable light-emitting diode (LED) array as a light source. Software was developed for data acquisition with a maximum of parameter variability and for background suppression. This approach allows the operation of the system even under daylight. Optical sensors showing analyte-specific changes of their luminescence decay time were tested and used for sensing pO2, pCO2, pH, and temperature. The luminophores employed are either platinum(II)-porphyrins or ruthenium(II)-polypyridyl complexes, contained in polymer films, and can be efficiently excited by blue LEDs. The decay times of the sensor films vary from 70 μs for the Pt(II)-porphyrins to several 100 ns for the Ru(II) complexes. In a typical application, 7 mm-diameter spots of the respective optical sensor films were placed at the bottom of the wells of microtiterplates. Thus, every well represents a separate calibration chamber with an integrated sensor element. Both luminescence intensity-based and time-resolved images of the sensor spots were evaluated and compared. The combination of optical sensor technology with time-resolved imaging allows a determination of the distribution of chemical or physical parameters in heterogeneous systems and is therefore a powerful tool for screening and mapping applications.

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.

Luminescence lifetime imaging with transparent oxygen optodes

The imaging of two-dimensional (2D) solute distributions with planar optodes has become an important tool in biological and medical research. The development of versatile and flexible imaging systems, that enable both luminescence intensity and lifetime imaging, has generated various applications of planar oxygen optodes. Most of the applied optodes however, were not transparent. They either contained scattering particles in the sensing layer for signal enhancement and/or an optical insulation to separate the signal from ambient light. Since the modular luminescence lifetime imaging system (MOLLI) enables luminescence lifetime imaging, we used transparent planar oxygen optodes to investigate simultaneously the 2D distribution of oxygen and the structure that causes this distribution. This is done by either using the luminescence intensity images or different spectral illumination for structural imaging and the luminescence lifetime images for oxygen distribution imaging. As the distribution of oxygen plays a key role at different spatial scales, we present results from applications of the transparent optodes to various biological systems: (a) to a coral sand sediment sample (macrolens application: resolution of approximately 50 mm per pixel); (b) to a lichen with cyanobacteria as symbionts (endoscope application: resolution of approximately 15‐62.5 mm per pixel) and (c) to a foraminifer with diatoms as symbionts (microscope application: resolution of approximately 3.8 mm per pixel). The results demonstrate the performance and some of the limits of the application of transparent optodes. Other possible fields of applications that are not restricted to marine environment are discussed. # 2001 Elsevier Science B.V. All rights reserved.

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.

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.

Oxygen flux fluorescence lifetime imaging

We describe a device capable of imaging distributions of surface PO2 as well as oxygen flux (e.g., into the human skin). Oxygen is monitored by a phase-angle-sensitive imaging technique using the effect of luminescence lifetime quenching of ruthenium complexes incorporated into a polymer layer system. The dyes are excited by modulated radiation of blue light-emitting diodes. The phase-shifted luminescence is detected by an intensified CCD camera. Several modulation techniques have been comparatively investigated. The obtained images correspond pixel-by-pixel to the local luminescence lifetimes, which are a direct measure for the distribution of surface PO2, or alternatively for the oxygen flux through a permeable diffusion barrier. For an illuminated sensor area of 10 cm2 and a spatial resolution of 1 mm at the sensor layer, the oxygen resolution is better than ΔPO2 = 0.3 torr in the absence of oxygen, and better than ΔPO2 = 4 torr at PO2= 160 torr.

FLOX—an oxygen-flux-measuring system using a phase-modulation method to evaluate the oxygen-dependent fluorescence lifetime

The direct relationship between the oxygen supply by skin blood flow and oxygen uptake (O 2 uptake) through the skin could be of importance for the diagnosis of circulatory disturbances and their consequences. A new measuring system has been developed to obtain simultaneously at three places the local O 2 uptake through the skin. It uses the principle of the O 2 -flux optode to measure the O 2 flux into the tissue. This luminescence-based-O 2 sensor has the well-known advantages (1) to be permeable for the analyte O 2 , (2) to be flexible to cover larger areas of the surface and (3) not to consume the analyte O 2 . To avoid the problems inherent to fluorescence intensity measurements the FLOX system (an oxygen-flux-measuring system) uses a phase-modulation measuring method to evaluate the oxygen-dependent fluorescence lifetime. The evaluation is based on the quadrature or incoherent envelope detection, which enables both phase angle (lifetime) and amplitude (intensity) to be received. The three sensor modules of the system consist of a light-emitting diode (LED) (λ peak = 470 nm) as light source, optical filters, a bifurcated glass fibre bundle to transport the light for excitation and emission, a photomultiplier tube as a detector and additional circuits. The main frequency generation of the modulation and reference signals is performed by a direct digital synthesizer (DDS) with dual output. The system is PC-based and works at modulation frequencies in the range of 5-900 kHz. Depending on the used indicator dye and the measuring purpose, the frequency is adjusted for the optimum phase angle range. The first O 2 -flux measurements on human skin with the multilayer O 2 -flux sensor and reproducibility measurements with the FLOX system prove the ability of the method.

Adaptation, test and in situ measurements with O2 microopt(r)odes on benthic landers

Oxygen microopt(r)odes have recently been introduced as an alternative to microelectrodes in the field of aquatic biology. We here describe adaptation, test results and first in situ measurements made with O2 microopt(r)odes on deep-sea benthic landers. This includes a detailed description of the sensors, the mechanical mounting, and the necessary measuring system. Hydrostatic pressure effects on the sensors and the optical penetrators are evaluated and discussed. Further, in situ micoopt(r)ode data obtained by a profiling lander (Profilur) and a benthic chamber lander (Elinor) are presented, discussed and compared to measurements obtained simultaneously by Clark type O2 microelectrodes. The obtained data demonstrated that opt(r)odes are a realistic and good alternative to electrodes for landers and other measuring platforms during deep-sea deployments.

Novel measuring system for oxygen micro-optodes based on a phase modulation technique

New fiber optic oxygen microsensors (microoptrodes) for use in aquatic environments have recently been developed as an alternative to commonly used CLark-type oxygen microelectrodes. The microoptrodes have the advantage of no oxygen consumption and no stirring sensitivity combined with a simple manufacturing process of the sensors. To avoid problems inherent to luminescence intensity measurements like photobleaching, signal dependency on the optical properties of the surrounding medium and system drifts, a novel measuring system was developed. This system uses a phase modulation method to evaluate a signal phase shift that is caused by the oxygen dependent luminescence lifetime. The measuring system is based on simple solid state technology. High reliability and low costs of the system can therefore be combined with the ability of miniaturization and low power consumption. The system consists of three units: 1) the microoptrode with the optical setup [glass fiber coupler, optical filters, lenses, light source (light emitting diode) and light detection (photon multiplier tube)], 2) the analogue signal processing unit, including a special phase detection module, and 3) the digital signal processing unit, a personal computer or a microcontroller for control of the measuring system, display and data storage. First measurements of oxygen depth profiles in sediments and biofilms at high levels of ambient light demonstrated the advantages of phase shift based O2 measurements as compared to intensity based measurements with microoptrodes.

IMAGING OF OXYGEN DYNAMICS WITHIN THE ENDOLITHIC ALGAL COMMUNITY OF THE MASSIVE CORAL PORITES LOBATA1

We used transparent planar oxygen optodes and a luminescence lifetime imaging system to map (at a pixel resolution of <200 μm) the two‐dimensional distribution of O2 within the skeleton of a Porites lobata colony. The O2 distribution was closely correlated to the distribution of the predominant endolithic microalga, Ostreobium quekettii Bornet et Flahault that formed a distinct green band inside the skeleton. Oxygen production followed the outline of the Ostreobium band, and photosynthetic O2 production was detected at only 0.2 μmol photons m−2 · s−1, while saturation occurred at ∼37 μmol photons m−2 · s−1. Oxygen levels varied from ∼60% to 0% air saturation in the illuminated section of the coral skeleton in comparison to the darkened section. The O2 production within the Ostreobium band was lower in the region below the upward facing surface of the coral and elevated on the sides. Oxygen consumption in darkness was also greatest within the Ostreobium zone, as well as in the white skeleton zone immediately below the corallites. The rate of O2 depletion was not constant within zones and between zones, showing pronounced heterogeneity in endolithic respiration. When the coral was placed in darkness after a period of illumination, O2 levels declined by 50% within 20 min and approached steady‐state after 40–50 min in darkness. Our study demonstrates the use of an important new tool in endolith photobiology and presents the first data of spatially resolved O2 concentration and its correlation to the physical structures and specific zones responsible for O2 production and consumption within the coral skeleton.