Rainer Uhl - Academia.edu (original) (raw)
Papers by Rainer Uhl
Journal of Bacteriology, 1989
The two retinal-containing photoreceptors of halobacteria, P480 and sensory rhodopsin, are formed... more The two retinal-containing photoreceptors of halobacteria, P480 and sensory rhodopsin, are formed constitutively and inducibly, respectively. Both photoreceptors are synthesized as apoproteins in cells with nicotine-inhibited retinal synthesis and are reconstituted as chromoproteins by the addition of all-trans retinal to cell membrane preparations. The decrease in photoreceptor-mediated photophobic response at the stationary growth phase of cells is not due to photoreceptor degradation but due to a deficiency of the signal transduction chain in the cell.
Journal of Microscopy, 2014
SummaryIn widefield fluorescence microscopy, images from all but very flat samples suffer from fl... more SummaryIn widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms ‘quasi‐confocal images’ can be derived from a given number of such phase‐images. Here, we present an alternative structured illumination microscopy approach, which employs two‐dimensional patterns instead of a one‐dimensional one. While in one‐dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two‐dimensional approach it is shifted at a single, pattern‐dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would r...
Cell Motility and the Cytoskeleton, 1990
Phototaxis of the unicellular alga Chlarnydomonas was studied with subsecond time resolution by u... more Phototaxis of the unicellular alga Chlarnydomonas was studied with subsecond time resolution by using a newly developed taxigraph. The taxigraph determines the cell density in a particular volume element of a cuvette by measuring the amount of scattered light originating from ...
Medical Laser Application, 2007
The goal was to develop a light-microscope platform concept, which allows the structural and func... more The goal was to develop a light-microscope platform concept, which allows the structural and functional characterization of live cells in microtiterplates with a speed, sensitivity and versatility unattainable so far. The goal was achieved by combining several novel technological concepts:•using mineral-cast as body material, thus having a 14x better vibration damping than with conventional metal-materials;•employing a model-based digital control for a voice coil focus drive. The drive features an excursion of 10mm with nanometer resolution and with minimal response times (1ms for a 1μm step and<500ms for a 10mm step);•applying galvanometer-scanning technology to follow a continuously moving sample during image acquisition, thus avoiding the usual stop & go;•implementing rapid (millisecond) switching of excitation wavelengths in the wavelength-range from 280 to 680nm;•automated dual emission image registration and•easy integration of environmental control.
Novel Optical Instrumentation for Biomedical Applications III, 2007
We have developed a novel platform-concept, which allows the construction of an automated upright... more We have developed a novel platform-concept, which allows the construction of an automated upright light-microscope-based slide-scanning device. In order to achieve maximal speed at maximal image quality we have paid special attention to a highly rigid, highly stiff construction. By using novel materials we have been able to achieve vibration-damping characteristics many times better than conventional approaches. We have combined
Biophotonics 2007: Optics in Life Science, 2007
The goal was to develop a light-microscope platform concept, which allows characterization of liv... more The goal was to develop a light-microscope platform concept, which allows characterization of live cells in microtiter plates or in live-cell slide chambers with a speed, sensitivity and versatility unattainable so far. The goal was achieved by combining several novel technological ...
Methods in Enzymology, 1982
Publisher Summary This chapter discusses the assays and characterization of Mg 2+ - adenosine tri... more Publisher Summary This chapter discusses the assays and characterization of Mg 2+ - adenosine triphosphatase (ATPase) in the rod outer segment (ROS) of vertebrate photoreceptors. It reports the existence of a specific ATPase system that undoubtedly resides in the photoreceptor outer segment and whose presence can be assayed by three distinct techniques: (1) conventional assay of ATPase activity measuring the amount of inorganic phosphate liberated, (2) light-scattering: “A D .” A large decrease in turbidity occurs when rod outer segments (ROS) are incubated in the presence of Mg 2+ and ATP in the dark, and (3) light-scattering: “A L .” A rapid further decrease in turditity occurs on flash illumination when ROS had been preincubated in the presence of Mg 2+ and ATP. ATP hydrolysis by ROS membranes was measured as release of inorganic phosphate, employing the method of Taussky and Schorr. When ROS fragments are incubated with both Mg 2+ and ATP in the dark, their turbidity is rapidly reduced.
Medical & Biological Engineering & Computing, 2007
In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilizat... more In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.
Journal of Biochemical and Biophysical Methods, 1984
A wide variety of biologically relevant chemical intermediates have been identified and character... more A wide variety of biologically relevant chemical intermediates have been identified and characterised by their spectral properties. When rapid kinetics, i.e. rapid changes in these spectral properties are studied, the equipment designed for these studies (flash photolysis-, T-jump apparatus) usually allows only the registration of intensity changes of the monitoring light beam at one particular wavelength. Quite frequently, however, particularly in biological systems, the reactions of interest are too complex to be fully understood using single wavelength techniques. We have therefore designed and built a flash photolysis apparatus which permits the simultaneous recording of absorbance changes at 32 wavelengths, freely selectable between 300 and 1000 nm, as well as changes in fluorescence, luminescence, birefringence and light scattering. The apparatus, which we have called Polychromatic Flash Photolysis Apparatus (PFPA), acquires up to 8000 difference spectra per second with an amplitude resolution of better than 0.0001 absorbance unit. Data acquisition and activation of an actinic xenon flash unit occurs under computer control. The same computer is responsible for data storage, handling and graphic display. This communication describes the PFPA, its performance, and, as a demonstration of its potential usefulness, its application to the measurement of the light driven photocycle of bacterial rhodopsin, the proton pumping protein of Halobacterium halobium.
Journal of Neuroscience Methods, 2011
2-Photon laser scanning microscopy (TPLSM) is often used for chronic in vivo studies. Small devia... more 2-Photon laser scanning microscopy (TPLSM) is often used for chronic in vivo studies. Small deviations in the sample orientation, however, make comparison of three-dimensional image stacks taken at different time-points challenging. When analysing changes of three-dimensional structures over time (4D imaging) this fundamental problem is one of the main limitations when complex structures are studied repetitively. We used an upright two-photon microscope complemented with a software-controlled stage-rotation instead of a conventional stage for chronic in vivo imaging in the brain of transgenic mouse models of Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease. Before every session an optimal imaging condition was successfully created by aligning the surface of the cranial window perfectly perpendicular to the laser beam. Deviations in the sample orientation between consecutive imaging sessions could be eliminated which improves conditions for chronic in vivo studies.
IEEE Transactions on Automation Science and Engineering, 2006
AbstractAddressing the increasing biomedical and pharma-cological interest in multiparametric sc... more AbstractAddressing the increasing biomedical and pharma-cological interest in multiparametric screening assays, a concept has been developed, which integrates multiparametric, bioelectric, and biochemical sensors for the analytical monitoring of intra-and extra ...
Biophysics of Structure and Mechanism, 1981
Biophysics of Structure and Mechanism, 1981
Biophysics of Structure and Mechanism, 1980
Reports on ATPase activity in rod outer segments (ROS) have been conflicting, mainly because it h... more Reports on ATPase activity in rod outer segments (ROS) have been conflicting, mainly because it has been very difficult to establish that the ATPase activity measured from ROS material actually resides in the ROS and not in small amounts Of contaminating material. Here we present evidence for the existence of a very specific Mg-ATPase system in ROS of both bovine and frog retinae. Its activity can be monitored in three ways:
Biophysics of Structure and Mechanism, 1980
A convenient way of studying rapidly occuring processes in biological systems is to monitor accom... more A convenient way of studying rapidly occuring processes in biological systems is to monitor accompanying spectral changes of intrinsic or externally applied optical probes. Unfortunately these spectral changes are quite frequently obscured by underlying changes in light scattering from the membrane material studied. Thus it becomes necessary to monitor rapid changes of the complete spectrum rather than its absorption changes at a single wavelength.
Biophysical Journal, 1991
Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlle... more Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself. The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 x longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a 3-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.
Journal of Bacteriology, 1989
The two retinal-containing photoreceptors of halobacteria, P480 and sensory rhodopsin, are formed... more The two retinal-containing photoreceptors of halobacteria, P480 and sensory rhodopsin, are formed constitutively and inducibly, respectively. Both photoreceptors are synthesized as apoproteins in cells with nicotine-inhibited retinal synthesis and are reconstituted as chromoproteins by the addition of all-trans retinal to cell membrane preparations. The decrease in photoreceptor-mediated photophobic response at the stationary growth phase of cells is not due to photoreceptor degradation but due to a deficiency of the signal transduction chain in the cell.
Journal of Microscopy, 2014
SummaryIn widefield fluorescence microscopy, images from all but very flat samples suffer from fl... more SummaryIn widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms ‘quasi‐confocal images’ can be derived from a given number of such phase‐images. Here, we present an alternative structured illumination microscopy approach, which employs two‐dimensional patterns instead of a one‐dimensional one. While in one‐dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two‐dimensional approach it is shifted at a single, pattern‐dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would r...
Cell Motility and the Cytoskeleton, 1990
Phototaxis of the unicellular alga Chlarnydomonas was studied with subsecond time resolution by u... more Phototaxis of the unicellular alga Chlarnydomonas was studied with subsecond time resolution by using a newly developed taxigraph. The taxigraph determines the cell density in a particular volume element of a cuvette by measuring the amount of scattered light originating from ...
Medical Laser Application, 2007
The goal was to develop a light-microscope platform concept, which allows the structural and func... more The goal was to develop a light-microscope platform concept, which allows the structural and functional characterization of live cells in microtiterplates with a speed, sensitivity and versatility unattainable so far. The goal was achieved by combining several novel technological concepts:•using mineral-cast as body material, thus having a 14x better vibration damping than with conventional metal-materials;•employing a model-based digital control for a voice coil focus drive. The drive features an excursion of 10mm with nanometer resolution and with minimal response times (1ms for a 1μm step and<500ms for a 10mm step);•applying galvanometer-scanning technology to follow a continuously moving sample during image acquisition, thus avoiding the usual stop & go;•implementing rapid (millisecond) switching of excitation wavelengths in the wavelength-range from 280 to 680nm;•automated dual emission image registration and•easy integration of environmental control.
Novel Optical Instrumentation for Biomedical Applications III, 2007
We have developed a novel platform-concept, which allows the construction of an automated upright... more We have developed a novel platform-concept, which allows the construction of an automated upright light-microscope-based slide-scanning device. In order to achieve maximal speed at maximal image quality we have paid special attention to a highly rigid, highly stiff construction. By using novel materials we have been able to achieve vibration-damping characteristics many times better than conventional approaches. We have combined
Biophotonics 2007: Optics in Life Science, 2007
The goal was to develop a light-microscope platform concept, which allows characterization of liv... more The goal was to develop a light-microscope platform concept, which allows characterization of live cells in microtiter plates or in live-cell slide chambers with a speed, sensitivity and versatility unattainable so far. The goal was achieved by combining several novel technological ...
Methods in Enzymology, 1982
Publisher Summary This chapter discusses the assays and characterization of Mg 2+ - adenosine tri... more Publisher Summary This chapter discusses the assays and characterization of Mg 2+ - adenosine triphosphatase (ATPase) in the rod outer segment (ROS) of vertebrate photoreceptors. It reports the existence of a specific ATPase system that undoubtedly resides in the photoreceptor outer segment and whose presence can be assayed by three distinct techniques: (1) conventional assay of ATPase activity measuring the amount of inorganic phosphate liberated, (2) light-scattering: “A D .” A large decrease in turbidity occurs when rod outer segments (ROS) are incubated in the presence of Mg 2+ and ATP in the dark, and (3) light-scattering: “A L .” A rapid further decrease in turditity occurs on flash illumination when ROS had been preincubated in the presence of Mg 2+ and ATP. ATP hydrolysis by ROS membranes was measured as release of inorganic phosphate, employing the method of Taussky and Schorr. When ROS fragments are incubated with both Mg 2+ and ATP in the dark, their turbidity is rapidly reduced.
Medical & Biological Engineering & Computing, 2007
In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilizat... more In research, pharmacologic drug-screening and medical diagnostics, the trend towards the utilization of functional assays using living cells is persisting. Research groups working with living cells are confronted with the problem, that common endpoint measurement methods are not able to map dynamic changes. With consideration of time as a further dimension, the dynamic and networked molecular processes of cells in culture can be monitored. These processes can be investigated by measuring several extracellular parameters. This paper describes a high-content system that provides real-time monitoring data of cell parameters (metabolic and morphological alterations), e.g., upon treatment with drug compounds. Accessible are acidification rates, the oxygen consumption and changes in adhesion forces within 24 cell cultures in parallel. Addressing the rising interest in biomedical and pharmacological high-content screening assays, a concept has been developed, which integrates multi-parametric sensor readout, automated imaging and probe handling into a single embedded platform. A life-maintenance system keeps important environmental parameters (gas, humidity, sterility, temperature) constant.
Journal of Biochemical and Biophysical Methods, 1984
A wide variety of biologically relevant chemical intermediates have been identified and character... more A wide variety of biologically relevant chemical intermediates have been identified and characterised by their spectral properties. When rapid kinetics, i.e. rapid changes in these spectral properties are studied, the equipment designed for these studies (flash photolysis-, T-jump apparatus) usually allows only the registration of intensity changes of the monitoring light beam at one particular wavelength. Quite frequently, however, particularly in biological systems, the reactions of interest are too complex to be fully understood using single wavelength techniques. We have therefore designed and built a flash photolysis apparatus which permits the simultaneous recording of absorbance changes at 32 wavelengths, freely selectable between 300 and 1000 nm, as well as changes in fluorescence, luminescence, birefringence and light scattering. The apparatus, which we have called Polychromatic Flash Photolysis Apparatus (PFPA), acquires up to 8000 difference spectra per second with an amplitude resolution of better than 0.0001 absorbance unit. Data acquisition and activation of an actinic xenon flash unit occurs under computer control. The same computer is responsible for data storage, handling and graphic display. This communication describes the PFPA, its performance, and, as a demonstration of its potential usefulness, its application to the measurement of the light driven photocycle of bacterial rhodopsin, the proton pumping protein of Halobacterium halobium.
Journal of Neuroscience Methods, 2011
2-Photon laser scanning microscopy (TPLSM) is often used for chronic in vivo studies. Small devia... more 2-Photon laser scanning microscopy (TPLSM) is often used for chronic in vivo studies. Small deviations in the sample orientation, however, make comparison of three-dimensional image stacks taken at different time-points challenging. When analysing changes of three-dimensional structures over time (4D imaging) this fundamental problem is one of the main limitations when complex structures are studied repetitively. We used an upright two-photon microscope complemented with a software-controlled stage-rotation instead of a conventional stage for chronic in vivo imaging in the brain of transgenic mouse models of Alzheimer&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease. Before every session an optimal imaging condition was successfully created by aligning the surface of the cranial window perfectly perpendicular to the laser beam. Deviations in the sample orientation between consecutive imaging sessions could be eliminated which improves conditions for chronic in vivo studies.
IEEE Transactions on Automation Science and Engineering, 2006
AbstractAddressing the increasing biomedical and pharma-cological interest in multiparametric sc... more AbstractAddressing the increasing biomedical and pharma-cological interest in multiparametric screening assays, a concept has been developed, which integrates multiparametric, bioelectric, and biochemical sensors for the analytical monitoring of intra-and extra ...
Biophysics of Structure and Mechanism, 1981
Biophysics of Structure and Mechanism, 1981
Biophysics of Structure and Mechanism, 1980
Reports on ATPase activity in rod outer segments (ROS) have been conflicting, mainly because it h... more Reports on ATPase activity in rod outer segments (ROS) have been conflicting, mainly because it has been very difficult to establish that the ATPase activity measured from ROS material actually resides in the ROS and not in small amounts Of contaminating material. Here we present evidence for the existence of a very specific Mg-ATPase system in ROS of both bovine and frog retinae. Its activity can be monitored in three ways:
Biophysics of Structure and Mechanism, 1980
A convenient way of studying rapidly occuring processes in biological systems is to monitor accom... more A convenient way of studying rapidly occuring processes in biological systems is to monitor accompanying spectral changes of intrinsic or externally applied optical probes. Unfortunately these spectral changes are quite frequently obscured by underlying changes in light scattering from the membrane material studied. Thus it becomes necessary to monitor rapid changes of the complete spectrum rather than its absorption changes at a single wavelength.
Biophysical Journal, 1991
Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlle... more Orientation of the green alga Chlamydomonas in light (phototaxis and stop responses) is controlled by a visual system with a rhodopsin as the functional photoreceptor. Here, we present evidence that in Chlamydomonas wild-type cells all-trans retinal is the predominant isomer and that it is present in amounts similar to that of the rhodopsin itself. The ability of different retinal isomers and analog compounds to restore photosensitivity in blind Chlamydomonas cells (strain CC2359) was tested by means of flash-induced light scattering transients or by measuring phototaxis in a taxigraph. All-trans retinal reconstitutes behavioral light responses within one minute, whereas cis-isomers require at least 50 x longer incubation times, suggesting that the retinal binding site is specific for all-trans retinal. Experiments with 13-demethyl(dm)-retinal and short-chained analogs reveal that only chromophores with a 3-methyl group and at least three double bonds in conjugation with the aldehyde mediate function. Because neither 13-dm-retinal, nor 9,12-phenylretinal restores a functional rhodopsin, a trans/13-cis isomerisation seems to take place in the course of the activation mechanism. We conclude that with respect to its chromophore, Chlamydomonas rhodopsin bears a closer resemblence to bacterial rhodopsins than to visual rhodopsins of higher animals.