13443
2012
eng
e38098
5
7
article
1
2016-06-06
--
--
Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples
Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 \(\mu\)mx50\(\mu\)mx2.5\(\mu\)m. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.
PLoS One
10.1371/journal.pone.0038098
urn:nbn:de:bvb:20-opus-134434
PLoS ONE 7(5): e38098. doi:10.1371/journal.pone.0038098
Siddharth Nanguneri
Benjamin Flottmann
Heinz Horstmann
Mike Heilemann
Thomas Kuner
eng
uncontrolled
architecture
eng
uncontrolled
rat calyx
eng
uncontrolled
in-vivo
eng
uncontrolled
microscopy
eng
uncontrolled
resolution
eng
uncontrolled
proteins
eng
uncontrolled
transmission
eng
uncontrolled
ultrastructure
eng
uncontrolled
reconstruction
eng
uncontrolled
localization
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13443/Nanguneri_PLoSOne.pdf
13123
2013
eng
e1003198
2
9
article
1
2016-04-01
--
--
Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail
The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env\((\Delta CT)\) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.
PLoS Pathogens
10.1371/journal.ppat.1003198
urn:nbn:de:bvb:20-opus-131235
PLoS Pathogens 9(2): e1003198. doi:10.1371/journal.ppat.1003198
Walter Muranyi
Sebastian Malkusch
Barbara Müller
Mike Heilemann
Hans-Georg Kräusslich
eng
uncontrolled
ENV
eng
uncontrolled
fluorescent-probes
eng
uncontrolled
type-1 matrix
eng
uncontrolled
glycoprotein incorporation
eng
uncontrolled
GP41 cytoplasmic tail
eng
uncontrolled
human immunodeficiency virus
eng
uncontrolled
cellular proteins
eng
uncontrolled
plasma membrane
eng
uncontrolled
virions
eng
uncontrolled
particles
Krankheiten
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13123/103_Murany_Plos_Pathogens.pdf
12183
2013
eng
6
6
article
1
2015-11-11
--
--
Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells
Background: The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane.
Results: To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding.
Conclusions: Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.
BMC Biophysics
10.1186/2046-1682-6-6
2046-1682
urn:nbn:de:bvb:20-opus-121835
BMC Biophysics 2013, 6:6. doi:10.1186/2046-1682-6-6
Mariana S. Dietz
Daniel Hasse
Davide M. Ferraris
Antonia Göhler
Hartmut H. Niemann
Mike Heilemann
eng
uncontrolled
single-molecule photobleaching
eng
uncontrolled
fluorescence correlation spectroscopy
eng
uncontrolled
fluorescence
eng
uncontrolled
EGF receptor
eng
uncontrolled
rat hepatocytes
eng
uncontrolled
structural insights
eng
uncontrolled
Scatter factor
eng
uncontrolled
SEMA domain
eng
uncontrolled
hepatocyte-growth-factor
eng
uncontrolled
invasion protein-INLB
eng
uncontrolled
listeria-monocytogenes
eng
uncontrolled
tyrosine kinase
eng
uncontrolled
living cells
eng
uncontrolled
dimerization
eng
uncontrolled
MET receptor
eng
uncontrolled
Signal transduction
Physiologie und verwandte Themen
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/12183/003_Dietz_BMC_Biophysics.pdf
13096
2013
eng
e64023
5
8
article
1
2016-03-31
--
--
Simple Method for Sub-Diffraction Resolution Imaging of Cellular Structures on Standard Confocal Microscopes by Three-Photon Absorption of Quantum Dots
This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.
PLoS ONE
10.1371/journal.pone.0064023
urn:nbn:de:bvb:20-opus-130963
PLoS ONE 8(5): e64023. doi:10.1371/journal.pone.0064023
Anje Sporbert
Zoltan Cseresnyes
Meike Heidbreder
Petra Domaing
Stefan Hauser
Barbara Kaltschmidt
Christian Kaltschmidt
Mike Heilemann
Darius Widera
eng
uncontrolled
HIV
eng
uncontrolled
stem-cell
eng
uncontrolled
infection
Medizin und Gesundheit
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13096/083_Sporbert_Plos_One.pdf
13468
2012
eng
120078
2
article
1
2016-06-10
--
--
Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast
The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.
Open Biology
10.1098/rsob.120078
urn:nbn:de:bvb:20-opus-134682
Open Biology 2: 120078. doi:10.1098/rsob.120078
David Lando
Ulrike Endesfelder
Harald Berger
Lakxmi Subramanian
Paul D. Dunne
James McColl
David Klenerman
Antony M. Carr
Markus Sauer
Robin C. Allshire
Mike Heilemann
Ernest D. Laue
eng
uncontrolled
nucleosome
eng
uncontrolled
fission yeast
eng
uncontrolled
identification
eng
uncontrolled
propagation
eng
uncontrolled
CSE4, CENP-A
eng
uncontrolled
CENP-A
eng
uncontrolled
schizosaccaromyces-pombe
eng
uncontrolled
fluorescent protein
eng
uncontrolled
centomeres
eng
uncontrolled
superresolution
eng
uncontrolled
chromatin
eng
uncontrolled
centromere
eng
uncontrolled
ingle-molecule microscopy
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13468/Lando_OpenBiology.pdf
6365
2011
eng
article
1
2013-01-16
--
--
Chemically Induced Photoswitching of Fluorescent Probes - A General Concept for Super-Resolution Microscopy
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
urn:nbn:de:bvb:20-opus-74896
7489
In: Molecules (2011) 16, 3106-3118; doi:10.3390/molecules16043106
Ulrike Endesfelder
Sebastian Malkusch
Benjamin Flottmann
Justine Mondry
Piotr Liguzinski
Peter J. Verveer
Mike Heilemann
deu
swd
Super-Resolution Microscopy
eng
uncontrolled
photoswitchable organic fluorophores
eng
uncontrolled
fluorescent proteins
eng
uncontrolled
super-resolution
eng
uncontrolled
PALM
eng
uncontrolled
dSTORM
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/6365/Heilemann_molecules_16_03106_v2.pdf
13408
2011
eng
3106-3118
4
16
article
1
2016-05-27
--
--
Chemically Induced Photoswitching of Fluorescent Probes - A General Concept for Super-Resolution Microscopy
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
Molecules
10.3390/molecules16043106
urn:nbn:de:bvb:20-opus-134080
Molecules 2011, 16, 3106-3118; doi:10.3390/molecules16043106
Ulrike Endesfelder
Sebastian Malkusch
Benjamin Flottmann
Justine Mondry
Piotr Liguzinski
Peter J. Verveer
Mike Heilemann
eng
uncontrolled
Photoactivated localization microscopy
eng
uncontrolled
Fusion proteins
eng
uncontrolled
Molecules
eng
uncontrolled
Patterns
eng
uncontrolled
Switch
eng
uncontrolled
Limit
eng
uncontrolled
Time
eng
uncontrolled
photoswitchable organic fluorophores
eng
uncontrolled
fluorescent proteins
eng
uncontrolled
super-resolution
eng
uncontrolled
PALM
eng
uncontrolled
dSTORM
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13408/024_Endesfelder_molecules.pdf
13353
2011
eng
e22007
7
6
article
1
2016-05-13
--
--
A SNAP-Tagged Derivative of HIV-1-A Versatile Tool to Study Virus-Cell Interactions
Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.
PLoS ONE
10.1371/journal.pone.0022007
urn:nbn:de:bvb:20-opus-133534
PLoS ONE 6(7): e22007. doi:10.1371/journal.pone.0022007
false
true
Manon Eckhardt
Maria Anders
Walter Muranyi
Mike Heilemann
Jacomine Krijnse-Locker
Barbara Müller
eng
uncontrolled
Human-immunodeficiency-virus
eng
uncontrolled
Fusion proteins
eng
uncontrolled
Live cells
eng
uncontrolled
Fluorescence microscopy
eng
uncontrolled
Stimulated-emission
eng
uncontrolled
Plasma-membrane
eng
uncontrolled
Living cells
eng
uncontrolled
Real-time
eng
uncontrolled
TYPE-1
eng
uncontrolled
GAG
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/13353/004_Eckhardt_Plos-ONE.pdf