TY  - JOUR
A1  - Lando, David
A1  - Endesfelder, Ulrike
A1  - Berger, Harald
A1  - Subramanian, Lakxmi
A1  - Dunne, Paul D.
A1  - McColl, James
A1  - Klenerman, David
A1  - Carr, Antony M.
A1  - Sauer, Markus
A1  - Allshire, Robin C.
A1  - Heilemann, Mike
A1  - Laue, Ernest D.
T1  - Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast
JF  - Open Biology
N2  - 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.
KW  - nucleosome
KW  - fission yeast
KW  - identification
KW  - propagation
KW  - CSE4, CENP-A
KW  - CENP-A
KW  - schizosaccaromyces-pombe
KW  - fluorescent protein
KW  - centomeres
KW  - superresolution
KW  - chromatin
KW  - centromere
KW  - ingle-molecule microscopy
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134682
VL  - 2
IS  - 120078
ER  - 
TY  - JOUR
A1  - Nanguneri, Siddharth
A1  - Flottmann, Benjamin
A1  - Horstmann, Heinz
A1  - Heilemann, Mike
A1  - Kuner, Thomas
T1  - Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples
JF  - PLoS One
N2  - 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.
KW  - architecture
KW  - rat calyx
KW  - in-vivo
KW  - microscopy
KW  - resolution
KW  - proteins
KW  - transmission
KW  - ultrastructure
KW  - reconstruction
KW  - localization
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134434
VL  - 7
IS  - 5
ER  -