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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.
Background: Novel microscopic techniques which bypass the resolution limit in light microscopy are becoming routinely established today. The higher spatial resolution of super-resolution microscopy techniques demands for precise correction of drift, spectral and spatial offset of images recorded at different axial planes.
Methods: We employ a hydrophilic gel matrix for super-resolution microscopy of cellular structures. The matrix allows distributing fiducial markers in 3D, and using these for drift correction and multi-channel registration. We demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores at different axial planes. We calculate a correction matrix for each spectral channel, correct for drift, spectral and spatial offset in 3D.
Results and discussion: We demonstrate single-molecule super-resolution microscopy with photoswitchable fluorophores in a hydrophilic gel matrix. We distribute multi-color fiducial markers in the gel matrix and correct for drift and register multiple imaging channels. We perform two-color super-resolution imaging of click-labeled DNA and histone H2B in different axial planes, and demonstrate the quality of drift correction and channel registration quantitatively. This approach delivers robust microscopic data which is a prerequisite for data interpretation.
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.
Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.
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(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread . 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.