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Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of... more Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of-focus fluorescence background deteriorates the illumination pattern and the reconstructed images suffer from influence of noise. We present a combination of structured illumination microscopy with line scanning. This technique reduces the out-of-focus fluorescence background, which improves the modulation and the quality of the illumination pattern and therefore facilitates the reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen's inner structure.
PloS one, 2015
Diatoms are unicellular algae of crucial importance as they belong to the main primary producers ... more Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of protein...
Journal of chemical ecology, Jan 14, 2015
Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous inse... more Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous insects. The spectrum of ingested and digested chlorophyll metabolites compares well to the pattern of early chlorophyll-degradation products in senescent plants. Intact chlorophyll is rapidly degraded by proteins in the front- and midgut. Unlike plants, insects convert both chlorophyll a and b into the corresponding catabolites. MALDI-TOF/MS imaging allowed monitoring the distribution of the chlorophyll catabolites along the gut of Spodoptera littoralis larvae. The chlorophyll degradation in the fore- and mid-gut is strongly pH dependent, and requires alkaline conditions. Using LC-MS/MS analysis we identified a lipocalin-type protein in the intestinal fluid of S. littoralis homolog to the chlorophyllide a binding protein from Bombyx mori. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes are covered with the chlorophyllide binding protein tig...
Optics express, Jan 23, 2015
We have developed an imaging system for 3D time-lapse polarization microscopy of living biologica... more We have developed an imaging system for 3D time-lapse polarization microscopy of living biological samples. Polarization imaging reveals the position, alignment and orientation of submicroscopic features in label-free as well as fluorescently labeled specimens. Optical anisotropies are calculated from a series of images where the sample is illuminated by light of different polarization states. Due to the number of images necessary to collect both multiple polarization states and multiple focal planes, 3D polarization imaging is most often prohibitively slow. Our MF-PolScope system employs multifocus optics to form an instantaneous 3D image of up to 25 simultaneous focal-planes. We describe this optical system and show examples of 3D multi-focus polarization imaging of biological samples, including a protein assembly study in budding yeast cells.
Optik - International Journal for Light and Electron Optics, 2001
From Principles to Biological Applications, 2013
Briefings in functional genomics & proteomics, 2006
Fluorescent imaging microscopy has been an essential tool for biologists over many years, especia... more Fluorescent imaging microscopy has been an essential tool for biologists over many years, especially after the discovery of the green fluorescent protein and the possibility of tagging virtually every protein with it. In recent years dramatic enhancement of the level of detail at which a fluorescing structure of interest can be imaged have been achieved. We review classical and new developments in high-resolution microscopy, and describe how these methods have been used in biological research. Classical methods include widefield and confocal microscopy whereas novel approaches range from linear methods such as 4Pi, I(5) and structured illumination microscopy to non-linear schemes such as stimulated emission depletion and saturated structured illumination. Localization based approaches (e.g. PALM and STORM), near-field methods and total internal refraction microscopy are also discussed. As the terms 'resolution', 'sensitivity', 'sampling' and 'precision...
RNA (New York, N.Y.), 2005
mRNP remodeling events required for the transition of an mRNA from active translation to degradat... more mRNP remodeling events required for the transition of an mRNA from active translation to degradation are currently poorly understood. We identified protein factors potentially involved in this transition, which are present in mammalian P bodies, cytoplasmic foci enriched in 5' --> 3' mRNA degrading enzymes. We demonstrate that human P bodies contain the cap-binding protein eIF4E and the related factor eIF4E-transporter (eIF4E-T), suggesting novel roles for these proteins in targeting mRNAs for 5' --> 3' degradation. Furthermore, fluorescence resonance energy transfer (FRET) studies indicate that eIF4E interacts with eIF4E-T and the putative DEAD box helicase rck/p54 in the P bodies in vivo. RNAi-mediated knockdowns revealed that a subset of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and eIF4E in P bodies. In addition, treatment of HeLa cells with cycloheximide, which inhibits translation, revealed ...
ABSTRACT In the past decade revolutionary advances have been made in the field of microscopy imag... more ABSTRACT In the past decade revolutionary advances have been made in the field of microscopy imaging. One such method is based on transforming conventionally unresolvable details into measurable patterns with the help of an effect most people have already personally experienced: the Moir effect. If two fine periodic patterns overlap, coarse patterns emerge. This is typically seen on a finely weaved curtain folding back onto itself. Another example is fast moving coarse patterns on both fences of a bridge above a motorway, when approaching it with the car. The microscopy method of structured illumination utilizes this effect by projecting a fine grating onto the sample and imaging the resulting coarser Moir patterns containing the information about invisibly fine sample detail. With the help of computer reconstruction based on several such Moire images, a high‐resolution image of the sample can then be assembled. Another way to obtain a high‐resolution map of the sample is to utilize the blinking behavior inherent in most molecules, used to stain the sample. Recent methodological advances (Cox et al., Nature Methods 9, 195‐200, 2012) enable us to create pointillist high‐resolution maps of molecular locations in a living biological sample, even if in each of the required many individual images, these molecules are not individually discernible. Examples will be shown as a film of a cell at 30 millionths of millimeter resolution and 6 seconds between the individual movie frames.
Optics express, Jan 6, 2014
We propose non-negative matrix factorisation with iterative restarts (iNMF) to model a noisy data... more We propose non-negative matrix factorisation with iterative restarts (iNMF) to model a noisy dataset of highly overlapping fluorophores with intermittent intensities. We can recover high-resolution images of individual sources from the optimised model, despite their high mutual overlap in the original data. Each source can have an arbitrary, unknown shape of the PSF and blinking behaviour. This allows us to use quantum dots as bright and stable fluorophores for localisation microscopy. We compare the iNMF results to CSSTORM, 3B and bSOFI. iNMF shows superior performance in the challenging task of super-resolution imaging using quantum dots. We can also retrieve axial localisation of the sources from the shape of the recovered PSF.
Optical Biopsies and Microscopic Techniques III, 1999
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues V, 2007
Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of... more Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of-focus fluorescence background deteriorates the illumination pattern and the reconstructed images suffer from influence of noise. We present a combination of structured illumination microscopy with line scanning. This technique reduces the out-of-focus fluorescence background, which improves the modulation and the quality of the illumination pattern and therefore facilitates the reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s inner structure.
PloS one, 2015
Diatoms are unicellular algae of crucial importance as they belong to the main primary producers ... more Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of protein...
Journal of chemical ecology, Jan 14, 2015
Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous inse... more Chlorophylls, the most prominent natural pigments, are part of the daily diet of herbivorous insects. The spectrum of ingested and digested chlorophyll metabolites compares well to the pattern of early chlorophyll-degradation products in senescent plants. Intact chlorophyll is rapidly degraded by proteins in the front- and midgut. Unlike plants, insects convert both chlorophyll a and b into the corresponding catabolites. MALDI-TOF/MS imaging allowed monitoring the distribution of the chlorophyll catabolites along the gut of Spodoptera littoralis larvae. The chlorophyll degradation in the fore- and mid-gut is strongly pH dependent, and requires alkaline conditions. Using LC-MS/MS analysis we identified a lipocalin-type protein in the intestinal fluid of S. littoralis homolog to the chlorophyllide a binding protein from Bombyx mori. Widefield and high-resolution autofluorescence microscopy revealed that the brush border membranes are covered with the chlorophyllide binding protein tig...
Optics express, Jan 23, 2015
We have developed an imaging system for 3D time-lapse polarization microscopy of living biologica... more We have developed an imaging system for 3D time-lapse polarization microscopy of living biological samples. Polarization imaging reveals the position, alignment and orientation of submicroscopic features in label-free as well as fluorescently labeled specimens. Optical anisotropies are calculated from a series of images where the sample is illuminated by light of different polarization states. Due to the number of images necessary to collect both multiple polarization states and multiple focal planes, 3D polarization imaging is most often prohibitively slow. Our MF-PolScope system employs multifocus optics to form an instantaneous 3D image of up to 25 simultaneous focal-planes. We describe this optical system and show examples of 3D multi-focus polarization imaging of biological samples, including a protein assembly study in budding yeast cells.
Optik - International Journal for Light and Electron Optics, 2001
From Principles to Biological Applications, 2013
Briefings in functional genomics & proteomics, 2006
Fluorescent imaging microscopy has been an essential tool for biologists over many years, especia... more Fluorescent imaging microscopy has been an essential tool for biologists over many years, especially after the discovery of the green fluorescent protein and the possibility of tagging virtually every protein with it. In recent years dramatic enhancement of the level of detail at which a fluorescing structure of interest can be imaged have been achieved. We review classical and new developments in high-resolution microscopy, and describe how these methods have been used in biological research. Classical methods include widefield and confocal microscopy whereas novel approaches range from linear methods such as 4Pi, I(5) and structured illumination microscopy to non-linear schemes such as stimulated emission depletion and saturated structured illumination. Localization based approaches (e.g. PALM and STORM), near-field methods and total internal refraction microscopy are also discussed. As the terms 'resolution', 'sensitivity', 'sampling' and 'precision...
RNA (New York, N.Y.), 2005
mRNP remodeling events required for the transition of an mRNA from active translation to degradat... more mRNP remodeling events required for the transition of an mRNA from active translation to degradation are currently poorly understood. We identified protein factors potentially involved in this transition, which are present in mammalian P bodies, cytoplasmic foci enriched in 5' --> 3' mRNA degrading enzymes. We demonstrate that human P bodies contain the cap-binding protein eIF4E and the related factor eIF4E-transporter (eIF4E-T), suggesting novel roles for these proteins in targeting mRNAs for 5' --> 3' degradation. Furthermore, fluorescence resonance energy transfer (FRET) studies indicate that eIF4E interacts with eIF4E-T and the putative DEAD box helicase rck/p54 in the P bodies in vivo. RNAi-mediated knockdowns revealed that a subset of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and eIF4E in P bodies. In addition, treatment of HeLa cells with cycloheximide, which inhibits translation, revealed ...
ABSTRACT In the past decade revolutionary advances have been made in the field of microscopy imag... more ABSTRACT In the past decade revolutionary advances have been made in the field of microscopy imaging. One such method is based on transforming conventionally unresolvable details into measurable patterns with the help of an effect most people have already personally experienced: the Moir effect. If two fine periodic patterns overlap, coarse patterns emerge. This is typically seen on a finely weaved curtain folding back onto itself. Another example is fast moving coarse patterns on both fences of a bridge above a motorway, when approaching it with the car. The microscopy method of structured illumination utilizes this effect by projecting a fine grating onto the sample and imaging the resulting coarser Moir patterns containing the information about invisibly fine sample detail. With the help of computer reconstruction based on several such Moire images, a high‐resolution image of the sample can then be assembled. Another way to obtain a high‐resolution map of the sample is to utilize the blinking behavior inherent in most molecules, used to stain the sample. Recent methodological advances (Cox et al., Nature Methods 9, 195‐200, 2012) enable us to create pointillist high‐resolution maps of molecular locations in a living biological sample, even if in each of the required many individual images, these molecules are not individually discernible. Examples will be shown as a film of a cell at 30 millionths of millimeter resolution and 6 seconds between the individual movie frames.
Optics express, Jan 6, 2014
We propose non-negative matrix factorisation with iterative restarts (iNMF) to model a noisy data... more We propose non-negative matrix factorisation with iterative restarts (iNMF) to model a noisy dataset of highly overlapping fluorophores with intermittent intensities. We can recover high-resolution images of individual sources from the optimised model, despite their high mutual overlap in the original data. Each source can have an arbitrary, unknown shape of the PSF and blinking behaviour. This allows us to use quantum dots as bright and stable fluorophores for localisation microscopy. We compare the iNMF results to CSSTORM, 3B and bSOFI. iNMF shows superior performance in the challenging task of super-resolution imaging using quantum dots. We can also retrieve axial localisation of the sources from the shape of the recovered PSF.
Optical Biopsies and Microscopic Techniques III, 1999
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues V, 2007