Rainer Heintzmann - Profile on Academia.edu (original) (raw)

Papers by Rainer Heintzmann

Localisation microscopy using quantum dots

Line Scan - Structured Illumination Microscopy High-resolution imaging in thick fluorescent samples

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.

Multidimensional and Superresolution Microscopies

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

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...

Standardizing the resolution claims for coherent microscopy

Nature Photonics, 2016

PLOS ONE, 2015

The microscope image of a thick fluorescent sample taken at a given focal plane is plagued by out... more The microscope image of a thick fluorescent sample taken at a given focal plane is plagued by out-of-focus fluorescence and diffraction limited resolution. In this work, we show that a single slice of Structured Illumination Microscopy (two or three beam SIM) data can be processed to provide an image exhibiting tight sectioning and high transverse resolution. Our reconstruction algorithm is adapted from the blind-SIM technique which requires very little knowledge of the illumination patterns. It is thus able to deal with illumination distortions induced by the sample or illumination optics. We named this new algorithm thick slice blind-SIM because it models a three-dimensional sample even though only a single two-dimensional plane of focus was measured.

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...

MultiFocus Polarization Microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously

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

The formalism for incoherent imaging of two points separated by the Sparrow criterion is investig... more The formalism for incoherent imaging of two points separated by the Sparrow criterion is investigated in two and three dimensions. Two and three dimensional imaging enables us to distinguish between a single point object and a two point object better than from the one dimensional information. Projections of the two point object, performed by integrating along the axial direction, are shown to improve the observed resolution especially for low NA non confocal systems. There are however a number of different objects, whose images are shown to be virtually indistinguishable. Therefore, resolution measures should only be used in the presence of knowledge about the class of objects being imaged.

Band Limit and Appropriate Sampling in Microscopy

Cell Biology, 2006

Introduction to Optics and Photophysics

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&#3...

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 ...

Structured Illumination and the Analysis of Single Molecules in Cells

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.

Localisation microscopy with quantum dots using non-negative matrix factorisation

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

Confocal laser scanning fluorescence microscopy is presently being used widely in biomedical rese... more Confocal laser scanning fluorescence microscopy is presently being used widely in biomedical research. A severe limitation for its use is its often still insufficient resolution. In situ measurements in 3D conserved human cell nuclei showed that distance measurements between fluorescent targets located in the interior of such objects are limited to a resolution regime of appr. O.3 tm in lateral and appr. tm in axial direction. A technique to overcome these restrictions is the recently developed Spectral Precision Distance Microscopy (SPM). This approach allows the determination of distances between targets which cany different spectral signatures with high precision. In situ measurements revealed that the SPM approach allows the determination of distances in 3D intact cell nuclei with a "Resolution Equivalent" better than 50 nm. Here we present an improved chromatic shift calibration method for Spectral Precision Distance Microscopy. Furthermore, micro axial tomography allows the tilting of objects perpendicular to the optical axis; thus two objects can always be tilted in such a way that they can be recorded in the same focal plane. Therefore the error in distance determination is minimized. Here we present some preliminary data for the applicability of spectral precision distance microscopy (SPM) to micro axial microscopy.

Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues V, 2007

Three dimensional imaging provides high-content information from living intact biology, and can s... more Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

The EMBO Journal, 2004

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the asse... more Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.

PLoS ONE, 2009

It has been shown that thin metal-based films can at certain frequencies act as planar near-field... more It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such ''lenses'' is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness) that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP) in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping) of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallicalloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (T-matrix) calculations that were performed in the MatLab TM and MathCad TM environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close ( 10 nm) or too far ( 30 nm) from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub-wavelength grating structure (such as in the far-field superlens design previously proposed [1]) or a fast nearfield scanning probe, it could provide a means for fast fluorescent imaging with sub-diffraction limit resolution.

PLoS ONE, 2010

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods ... more Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of redconverted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow ,20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ,70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10-30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.