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1.
PLoS Pathog ; 9(3): e1003213, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23505371

ABSTRACT

The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.


Subject(s)
Protozoan Proteins/metabolism , Toxoplasma/metabolism , rab5 GTP-Binding Proteins/metabolism , Cell Line , Fibroblasts/parasitology , Humans , Organelles/metabolism , Protein Transport , Protozoan Proteins/genetics , Secretory Pathway
2.
Opt Express ; 20(5): 5225-36, 2012 Feb 27.
Article in English | MEDLINE | ID: mdl-22418329

ABSTRACT

In stimulated emission depletion (STED) nanoscopy the wavelength of the STED beam is usually tuned towards the red tail of the emission maximum of the fluorophore. Shifting the STED wavelength closer to the emission peak, i.e. towards the blue region, favorably increases the stimulated emission cross-section. However, this blue-shifting also increases the probability to excite fluorophores that have remained in their ground state, compromising the image contrast. Here we present a method to exploit the higher STED efficiency of blue-shifted STED beams while maintaining the contrast in the image. The method is exemplified by imaging immunolabeled features in mammalian cells with an up to 3-fold increased STED efficiency compared to that encountered in standard STED nanoscopy implementations.


Subject(s)
Image Enhancement/instrumentation , Microscopy, Fluorescence/instrumentation , Nanotechnology/instrumentation , Equipment Design , Equipment Failure Analysis
3.
Opt Express ; 19(9): 8066-72, 2011 Apr 25.
Article in English | MEDLINE | ID: mdl-21643055

ABSTRACT

We show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs. A spatial resolution of 40-50 nm is attained by pulsing a 660 nm DVD-diode. The efficacy of these low-cost STED microscopes in biological imaging is demonstrated by differentiating between clusters of the synaptic protein bassoon and transport vesicles in hippocampal neurons, based on the feature diameter. Our results facilitate the implementation of this all-molecular-transition based superresolution method in many applications ranging from nanoscale fluorescence imaging to nanoscale fluorescence sensing.


Subject(s)
Image Enhancement/instrumentation , Lasers, Semiconductor , Microscopy, Fluorescence/instrumentation , Nanotechnology/instrumentation , Equipment Design , Equipment Failure Analysis
4.
Opt Express ; 19(6): 5644-57, 2011 Mar 14.
Article in English | MEDLINE | ID: mdl-21445205

ABSTRACT

We report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable (fluorescence) state transitions induced by a spatially modulated light pattern. The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce. By switching fluorophores off only if there is an adjacent fluorescent feature to be recorded, the method reduces the number of state transitions as well as the average time a dye is forced to reside in an off-state. Thus, the photobleaching of the sample is reduced, while resolution and recording speed are preserved. The power of the method is exemplified by imaging immunolabeled glial cells with up to 8-fold reduced photobleaching.


Subject(s)
Nanotechnology/methods , Optics and Photonics/methods , Amyloid beta-Protein Precursor/metabolism , Animals , Cell Line, Tumor , Fluorescent Dyes/metabolism , Glial Fibrillary Acidic Protein/metabolism , Humans , Imaging, Three-Dimensional , Mice , Microspheres , Neurons/cytology , Neurons/metabolism , Photobleaching
5.
Nature ; 461(7267): 1069-70, 2009 Oct 22.
Article in English | MEDLINE | ID: mdl-19847257
6.
Nano Lett ; 9(9): 3323-9, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19634862

ABSTRACT

Charged nitrogen-vacancy (NV) color centers in diamond are excellent luminescence sources for far-field fluorescence nanoscopy by stimulated emission depletion (STED). Here we show that these photostable color centers can be visualized by STED using simple continuous-wave or high repetition pulsed lasers (76 MHz) at wavelengths >700 nm for STED. Furthermore, we show that NV centers can be imaged in three dimensions (3D) inside the diamond crystal and present single-photon signatures of single color centers recorded in high density samples, demonstrating a new recording scheme for STED and related far-field nanoscopy approaches. Finally, we exemplify the potential of using nanodiamonds containing NV centers as luminescence tags in STED microscopy. Our results offer new experimental avenues in nanooptics, nanotechnology, and the life sciences.


Subject(s)
Light , Nanoparticles/chemistry , Nitrogen/chemistry , Luminescence , Materials Testing , Microscopy, Fluorescence , Nanotechnology , Particle Size , Surface Properties
7.
Opt Express ; 16(13): 9614-21, 2008 Jun 23.
Article in English | MEDLINE | ID: mdl-18575529

ABSTRACT

We report on a straightforward yet powerful implementation of stimulated emission depletion (STED) fluorescence microscopy providing subdiffraction resolution in the far-field. Utilizing the same super-continuum pulsed laser source both for excitation and STED, this implementation of STED microscopy avoids elaborate preparations of laser pulses and conveniently provides multicolor imaging. Operating at pulse repetition rates around 1 MHz, it also affords reduced photobleaching rates by allowing the fluorophore to relax from excitable metastable dark states involved in photodegradation. The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30-50 nm in the focal plane, i.e. by a factor of 8-9 beyond the diffraction barrier.


Subject(s)
Equipment Design , Lasers , Lighting/instrumentation , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentation , Equipment Failure Analysis , Reproducibility of Results , Sensitivity and Specificity
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