TY  - JOUR
A1  - Proppert, Sven
A1  - Wolter, Steve
A1  - Holm, Thorge
A1  - Klein, Theresa
A1  - van de Linde, Sebastian
A1  - Sauer, Markus
T1  - Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging
JF  - Optics Express
N2  - In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.
KW  - three-dimensional microscopy
KW  - fluorescence microscopy
KW  - medical and biological imaging
KW  - superresolution
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119730
SN  - 1094-4087
VL  - 22
IS  - 9
ER  - 
TY  - JOUR
A1  - Laine, Romain F.
A1  - Albecka, Anna
A1  - van de Linde, Sebastian
A1  - Rees, Eric J.
A1  - Crump, Colin M.
A1  - Kaminski, Clemens F.
T1  - Structural analysis of herpes simplex virus by optical super-resolution imaging
JF  - Nature Communications
N2  - Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.
KW  - tegument protein pUL36
KW  - fluorescence microscopy
KW  - monoclonal antibodies
KW  - 3-dimensional structure
KW  - type-1
KW  - nuclear pore complex
KW  - reconstruction microscopy
KW  - localization microscopy
KW  - resolution
KW  - envelopment
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144623
VL  - 6
IS  - 5980
ER  - 
TY  - JOUR
A1  - Wäldchen, Sina
A1  - Lehmann, Julian
A1  - Klein, Teresa
A1  - van de Linde, Sebastian
A1  - Sauer, Markus
T1  - Light-induced cell damage in live-cell super-resolution microscopy
JF  - Scientific Reports
N2  - Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of similar to 1 kW cm\(^{-2}\) at 640 nm for several minutes, the maximum dose at 405 nm is only similar to 50 J cm\(^{-2}\), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
KW  - optical reconstruction microscopy
KW  - tag fusion proteins
KW  - localization microscopy
KW  - photodynamic therapy
KW  - diffraction limit
KW  - illumination microscopy
KW  - structured illumination
KW  - fluorescent probes
KW  - in vitro
KW  - dynamics
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145207
VL  - 5
IS  - 15348
ER  -