Light sheet microscopy has become an indispensable tool for fast, low phototoxicity volumetric imaging of biological samples; predominantly providing structural or analyte concentration data in its standard format. Fluorescence lifetime imaging microscopy (FLIM) provides functional contrast, but often at limited acquisition speeds and with complex implementation.
We have developed a digitally scanned light sheet microscope for fast 2-colour volumetric imaging with imaging performed in the frequency domain at 20MHz using the PCO.FLIM camera. The camera enables rapid acquisition of two phases simultaneously at 0 and 180 degrees and with a phase shift relative to the modulated square-wave excitation. Whilst this frequency domain method has been well known for decades, application to light-sheet imaging is novel and provides straightforward functional read-out of fluorescence resonance energy transfer for protein interaction studies deep inside intact specimens such as Zebrafish. We demonstrate application of fluorescence lifetime contrast using the fluorescent protein biosensors in both live Zebrafish and organoids in digitally scanned light sheet FLIM. We apply signal processing techniques to improve data analysis and visualization and discuss this alongside practical application of real-time volumetric imaging of live biological specimens.
The Pressure-Sensitive-Paint (PSP) measurement technique is based on the dependence of the intensity or decay time of its luminescence on the pressure, brought about by oxygen quenching. PSP is usually exited by light of an appropriate wavelength (e.g. UV-light) and its pressure dependent luminescence decay time or lifetime is detected by a camera system (CCD or CMOS). Two basic types of lifetime measurement exist: the first type is a time-domain lifetime method. For this method a pulsed light is used to excite the paint and the pressure dependent time constant is determined from the decay curve of luminescence intensity. The second type is a frequency-domain fluorescence lifetime imaging (FLIM) where sinusoidal modulated light is used to excite the paint and the PSP luminescence is simultaneously detected to calculate its pressure dependent phase shift and amplitude ratio. Based on UV-LEDs a light source has been designed which provides high intensity stable and low distorted sine-modulated light of constant amplitude which is essential for the accuracy of the presented method. The new light source is used to investigate the influence of frequency on pressure sensitivity of a PSP sample to optimize the system for application in transonic wind tunnel tests.
The luminescence lifetime as a beneficial analytical parameter is known for many years and is well described by a large variety of publications. Many instruments including 2D measuring systems with cameras have been developed and applied in the past years. However, since the current instrumentation to perform either time- or frequency-domain lifetime measurements is rather complex, new developments in CMOS image sensor technology have achieved to create new image sensors, which can efficiently be integrated into easier-to-handle luminescence lifetime measuring systems. The principle of these modulatable CMOS image sensors, while initially being designed for distance measurements, shows a clear analogy to frequency-domain FLIM measurements, which was proven by researchers [1, 2]. Based on this principle a new CMOS image sensor has been developed, integrated into a camera system and has been investigated within a research project. The image sensor has a resolution of 1024 × 1024 pixels with a 5.6 μm pitch and can be modulated up to 50 MHz. First measurements show an effective dynamic range of larger than 1:1024 (corresponding to 10 bit dynamic). The maximum frame rate is in the range of 90 frames/s in dual-tap mode, resulting in an effective lifetime image frame rate for realistic measurements of approximately 22 frames/s. The camera system pco.flim, featuring that image sensor, generates all required modulation signals from 5 kHz to 50 MHz (sinusoidal and rectangular). It performs advanced pixel correction to generate linear and high-quality images, while the basic lifetime image processing is done in the computer. The modulation frequency can be freely adjusted within the specified range. The characteristics of the camera systems are presented, and first results are discussed using different representations of the data like for example the phasor approach [3], which has been established to provide a more global view to pixelwise fluorescence lifetime data and compare time- and frequency-domain results. Based on these results and the experiences of the on-going tests, it can be expected, that the pco.flim will significantly ease the introduction of luminescence lifetime imaging systems to broader applications.
Due to the MOdular Luminescence Lifetime Imaging system (MOLLI)that has been developed within the last few years it is possible to use oxygen sensors that are not optically isolated. Transparent planar optodes and dispersed nano-optodes for the first time enable a direct optical link between the chemical parameter to be measured (oxygen as an example) and the structure that is responsible for the distribution of the chemical parameter (ascidian and corals as an example). Since the transparency as a principal quality of the optode allows to record structural images. The spatial resolution of the MOLLI imaging system is determined by a)the area size of the view field that is imaged onto the amount of pixels of the CCD-chip (640x480 in our case)and b)by the spatial limitations of the sensing layer. The latter means in case of the planar optode the thickness of the sensing layer and in case of dispersed nano-optodes the thickness of the excitation light field. We present biological applications of transparent planar optodes (thickness approximately equals 5-10 micrometers ) at two areal resolutions,a)50 micrometers /pixel and b)6 micrometers /pixel and one application of nano-optodes at 80 micrometers /pixel. The first application shows the oxygen production of endolithic cells that live in the skeleton of massive corals measured in a cut coral sample that was illuminated through the planar oxygen optode with defined light energy levels to follow the oxygen production of these coral symbionts. Finally the oxygen production and consumption of coral symbionts are shown by dispersed oxygen nano-optodes in the medium. The specific set-up for the latter experiment will be discussed with the future implication of possible 3D measurements. Although the results all come from biological applications from coral reef environments obviously the measuring system and the transparent sensors can be applied to a variety of environmental topics. At the moment similar optodes are under development for parameters like pH,CO2 and temperature.
A fiberoptical optode array for the in-situ measurement of ground air oxygen concentrations has been used in both, lab and field experiments to monitor subsurface oxygen consumption in a lignite mine tailing affected by acid mine drainage formation. The single sensors are constructed from plastic fibers (core diameter 1 mm) with an oxygen sensitive fluorescent dye film attached to the fiber tip. Measurements were performed with a commercially available oxygen measuring instrument (MICROX 1, PreSens, Regensburg, Germany) which had been modified for the use with 1 mm plastic fibers. The instrument evaluates the oxygen dependent change of the luminescence lifetime of an oxygen indicator using a phase modulation technique. First measurements show a strong oxygen consumption by pyrite oxidation indicated by a ground air oxygen concentration gradient pointing to a depth of approximately 6 m. The measurement of the pyrite depth distribution of the material confirms the assumption that the 40 year old tailing has been depyritized down to a depth of 6 m and that pyrite oxidation and acid mine drainage formation are still going on. Investigations will proceed in order to assess long-term sensor stability under strongly acid conditions.
An improved refractive index microsensor based on surface plasmon resonance (SPR) for fine scale measurements in aquatic environments is presented. Furthermore this sensor should serve as a platform for different applications. In the first place refractive index measurements in marine environments have been performed to characterize the light conditions around photosynthetically active organisms. By scalar irradiance microprobes the light intensity can be investigated at a spatial resolution of 100micrometers . The presented sensor for refractive index achieves a spatial resolution better than 1mm. It covers a range of 1.30 and 1.38 refractive index units (RIU) with an accuracy of 5x10-4 RIU. Due to this accuracy, the miniaturized geometry and the simple preparation the sensor can serve as a platform for chemo- and biosensors with a high spatial resolution and fast response. Therefor a suitable sensitive layer that converts the specific analyte concentration into a refractive index change can be deposited onto the gold surface. For that purpose swellable polymer microspheres are currently investigated. The sensor characteristics, measurement system, and applications are presented.
KEYWORDS: Oxygen, Digital signal processing, Temperature metrology, Sensors, Luminescence, Signal processing, Modulation, Fiber optics sensors, Fiber optics, Signal to noise ratio
A new signal processing approach for fiberoptical sensors, microoptodes, is presented. All signal generation and processing is completely based on a fast, low-cost DSP (Digital Signal Processor). This enables the implementation of new features such as a simultaneous multi-frequency measurement to resolve different analytical parameters in the luminescence signal. For instance, a hybrid sensor was applied to simultaneously sense the temperature and oxygen concentration, and the temperature information was used to compensate for the temperature effect on the oxygen measurement. As a further benefit, recent improvements in indicator chemistry and fiber tip preparation also yield a luminescence signal level high enough to be detected with a common photodiode instead of a photomultiplier tube. Consequently, the combination of small detectors and highly integrated DSPs enable portable, handheld measurement devices with very little calibration requirements.
A new refractive index microsensor based on surface plasmon resonance (SPR) for fine scale measurements in aquatic environments is presented. The local light conditions in marine sediments determine the activity of photosynthetic organisms. The light field can be investigated by scalar irradiance microprobes at a spatial resolution better than 100 micrometer but the refraction index still has to be assumed constant or measured by techniques with lower spatial resolution. It is well known from other microsensor measurements that the microenvironment strongly determines the metabolism of the organisms. Therefore we developed the new sensor to access the fine scale distribution of refraction index. A second parameter of interest is the salinity which can be assumed not to be constant in some biofilms. As the salinity is usually measured with a refractometer, we checked if we could measure salinity independent of the ambient optical conditions in the sediment with the new microsensor. The microsensor is formed by a multimode silica fiber where the plastic jacket and fiber cladding has been removed. The tip is then tapered and a gold layer is deposited. Due to the tapered geometry the diameter is decreased to achieve a higher spatial resolution for profiling applications. SPR is excited at the fiber tip by coupling polychromatic light into the fiber. The reflected light is measured with a spectrometer. A refractive index change of the fiber tips surrounding area causes a wavelength shift of the spectral intensity distribution. The effects of different tip geometries have been characterized and results are presented. An appropriate measuring system is proposed.
A new sensor head and imaging application with planar oxygen optodes is presented. It combines the versatility of the recently presented modular luminescence lifetime imaging system (MOLLI) and the oxygen measuring features of planar optodes to investigate the 2D-distribution of oxygen with a high spatial resolution. The marine sediments are settled by micro-organisms. They are characterized by steep gradients of solutes perpendicular to the interface. Oxygen as the most favorable electron acceptor plays an important role in these communities and exhibits steep gradients within distances of 100 micrometer to a couple of millimeters. Traditionally these gradients are commonly accessed by either oxygen microelectrodes or oxygen micro-optodes, that measure single oxygen depth profiles at spatial resolutions in the range of 50 - 5 micrometer. As the influences on the metabolism of these organisms are complex, profiles that are measured at one location exhibit a natural heterogeneity. To address this problem by using the potential of planar oxygen optodes first measurements with optodes fixed to an aquarium wall (Glud et al. 1996) based on luminescence intensity measurements have been published. They showed the large amount of information that can be gathered by this method. We further developed this approach by combining the planar optode with the capabilities of imaging fibers that form an endoscope. We developed a periscope type sensor head that can be independently applied in biological samples. It has an overall diameter of 2.5 mm and a calculated pixel resolution of 12 micrometer. The according measuring system compared to the published system has a different excitation light setup and corresponding endoscope optics to measure the lifetime based images. We present the adapted system, sensor head and the first results of an application.
Sediments, microbial mats, biofilms and other microbial communities are characterized by steep gradients of physical and chemical parameters. Fibre optical microsensors, microoptodes, that we developed over the last three years have become powerful tools to investigate and measure these parameters with a sufficient spatial resolution and with a minor disturbance of the micro-environment in natural systems.
Established sensors for fine scale measurements in natural environments are based on electrochemical measuring principles for e. g. oxygen and pH. The preparation of such electrochemical sensors is, however, a time consuming process.
Benthic aquatic environments like biofilms or sediments are often investigation by measuring profiles of chemical or physical parameters at a high spatial resolution (< 50 micrometers ). This is necessary to understand e.g. transport processes and the biogeochemistry of the sediment water interface. A variety of electrochemical and optical microsensors has been developed and used for this purpose. In most of these applications the temperature of the investigated biofilms or sediments is assumed to be constant. However measurements with thermocouples of an appr. diameter of 300 micrometers have shown that this is not always the case for illuminated shallow water sediments and biofilms. We developed new microoptodes for measuring temperature distributions at a high spatial (< 50 micrometers ) and thermal (< 0.2 degree(s)C) resolution in aquatic systems. The new sensors are based on a fluorophore that is well known for its application in oxygen sensing-Ruthenium(II)- tris-1,10-phenantroline. Demas et al. (1992) discussed the possible use of highly luminescent transition metal complexes as temperature indicators. We have approached this idea from our experiences with ruthenium complexes as oxygen indicators. The first realized sensor consists of a closed microcapillary filled with an indicator solution and in inserted tapered optical fiber. The principle uses the temperature dependence of the fluorescence lifetime in the solution. To keep the solution oxygen free an oxygen scavenger is added to it. The change of the lifetime is detected by a special measuring device that uses a phase modulation technique.
Traditionally microscale measurements of pH are based on potentiometric measurements with a pH glass microelectrode. The preparation of these electrodes is, however, very time consuming. We developed pH micro-optodes for use in seawater in the range of pH 7 - 9. The optodes are based on immobilized acid-base indicators, which change their color and/or fluorescence properties as a function of the pH. Various dyes were immobilized directly on the tip of a tapered optical fiber by different techniques. We then investigated these pH optodes with respect to response time, mechanical stability and calibration characteristics. Dependent on the optical properties of the indicator material we used different light emitting diodes (LEDs) as the light sources and either a photodiode or a photomultiplier as detector.
The amount of dissolved hydrogen sulfide is an important parameter in many environmental applications. Conventional methods for H2S detection depend on iodometric titration or spectroscopic measurements. Unfortunately these methods are not applicable for direct measurements in natural systems. A recently described method for the on-line detection of H2S is based on quenching of fluorescence of thioneine. The reaction between H2S and thioneine was described as reversible photo-reduction. This reaction was tested in order to design an optical microsensor for the measurement of H2S in sediments and other biological systems. We immobilized thioneine in several matrices and investigated these materials with respect to response time, mechanical stability, the influence of the excitation light and the reversibility. The sensing materials were deposited on the tip of optical fibers. The measuring system for the excitation and detection of the fluorescence consisted of a yellow light emitting diode, a fiber-optic coupler and a photomultiplier. The excitation light was intensity modulated to enable measurements in ambient light. Our results indicate that the thioneine based reaction scheme for H2S detection is not very suitable for use in a H2S optode due to lack of reversibility, long response times, and the need for regeneration of the sensor chemistry.
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.
We present a new fiber-optic oxygen microsensor based on dynamic luminescence quenching which was recently developed for measuring oxygen at high spatial resolution in aquatic sediments and biofilms. Micro-optrodes with a typical tip diameter of 20 to 50 micrometers were fabricated. The fabrication procedure is simple and guarantees a high reproducibility of the calibration curves. The micro-optrodes were characterized with respect to dynamic range, response time, storage, longterm stability, interferences, temperature dependence, photostability, and mechanical stability. A special LED based luminescence intensity measuring instrument was developed. It is battery operated and can be used for field measurements. The micro-optrodes were used to measure oxygen gradients in marine sediments. Comparative measurements were performed with oxgen microelectrodes. The first measurements have shown that oxygen micro-optrodes present a true alternative to existing electrochemical microsensors. Nevertheless, it is obvious, that the measurement of luminescence intensity of the indicator limits their practical application. Therefore a new setup was developed to make oxygen measurements with the luminescence lifetime as parameter.
The development and improvement of oxygen sensors is continuously matter of research because it is important to quantify oxygen concentrations in biological systems. The presented hybrid fiberoptical sensor combines the common advantages of fiber optic sensors e.g. flexible connection to the place of interest and potential separation between place of measurement and measuring device with the special advantage of the optical analysis that does not consume the analyte oxygen. The O2 sensing was tested by using (1) solutions with the dissolved oxygen indicator dye (tris(1,10-phenantroline) ruthenium (II) chloride hexahydrate), and (2) and O2 flux optode. The O2 flux optode consist of an oxygen permeable membrane (test membrane) combined with oxygen optodes to measure the pO2 differences across the membrane. The O2 flux can be calculated from the pO2 gradient across the test membranes and its permeability. The developed measuring systems `FLOX' detects the phase shift between the sinusoidally modulated excitation light and the resulting fluorescence of the indicator. The phase shift is caused by the fluorescence lifetime that depends on the oxygen concentration. Many investigations have shown that measurements of lifetime are preferable to measurements of intensity because of higher stability. The FLOX system uses a blue light emitting diode (LED, (lambda) peak equals 450 nm) as light source together with optical filters and a photo multiplier tube as detector. The coupling between the place of measurement and the FLOX is performed by a bifurcated fiber bundle in which single multimode fibers are statistically mixed. Its measuring end could be either immersed directly in an aqueous solution of the fluorophore or provided with a special sensor head for the measurement of the oxygen flux into a surface, for example into the surface of the human skin. First measurements demonstrated the applicability of the FLOX system to measure the O2 flux into human skin; for example an about 20 - 50% increase of the O2 flux after stop of the circulation could be clearly shown.
About 1-2% of the basal human O2-consumption is taken up by the skin surface. This amount varies depending on the local skin blood flow. A new sensing device, the O2-flux- optode, has been developed which should allow the measurement of local O2-uptake of the skin and thereby indirectly its local circulation. This luminescence-based-sensor (1) is permeable for the analyte O2, (2) is flexible for covering larger areas of the surface, and (3) does not consume the analyte O2. The new O2-flux-optode consists of an indicator layer with an embedded oxygen sensitive fluorescence dye and a test layer with known diffusion properties. If this sensor is applied to the skin, the pO2 between skin surface and test layer is measured. With defined (or measured) pO2 of the environment (e.g., air) the O2-flux (JO(2)) into the tissue can be determined from the pO2 gradient ((Delta) pO2) across the test layer and its material properties (C). This yields to the equation: JO(2) equals C * (Delta) pO2.
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