Meike Heidbreder

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

Live-cell super-resolution imaging with trimethoprim conjugates

The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at ∼20 nm resolution.

Schwann Cells Can Be Reprogrammed to Multipotency by Culture

Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions.

TNF-α influences the lateral dynamics of TNF receptor I in living cells

In mammalian cells, inflammation is mainly mediated by the binding of tumor necrosis factor alpha to tumor necrosis factor receptor 1. In this study, we investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy. Our single-molecule data indicates the presence of tumor necrosis factor receptor 1 with different mobilities in the plasma membrane, suggesting different molecular organizations. Cholesterol depletion led to a decrease of slow receptor species and a strong increase in the average diffusion coefficient. Moreover, as a consequence of tumor necrosis factor-alpha treatment, the mean diffusion coefficient moderately increased while its distribution narrowed. Based on our observation, we propose a refined mechanism on the structural arrangement and activation of tumor necrosis factor receptor 1 in the plasma membrane.

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons.

Abstract. Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12±0.05 to 0.61±0.03  μm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9±1.9 to 15±4.9  μm/s, whereas a velocity increase from 9±1.3 to 14±3  μm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.

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.

Signal Transduction through the Met Receptor Studied at the Single-Molecule Level

The human receptor tyrosine kinase Met and its ligand hepatocyte growth factor (HGF) are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration [1]. 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 (InlB) and causes human listeriosis [2]. Many of the mechanistic steps of Met activation, however, are still unclear.Here, we use single-molecule fluorescence microscopy techniques [3, 4] to unravel association and activation of Met receptor through its ligands, HGF and InlB. In particular, we use techniques which provide high temporal and spatial resolution and excellent statistics to study both the spatial distribution as well as the dynamics of Met receptor in eukaryotic cells. In addition, we investigate interactions with membrane microdomains.[1] Birchmeier C.; Birchmeier W.; Gherardi E.; Vande Woude G.F. (2003) Met, metastasis, motility and more. Molecular Cell Biology, 4, 915-925.[2] Shen Y.; Naujokas M.; Park M.; Ireton K. (2000) InlB-Dependent Internalization of Listeria is mediated by the Met Receptor Tyrosine Kinase. Cell, 103, 501-510.[3] Hohlbein, J.; Gryte, K.; Heilemann, M. & Kapanidis, A. N. (2010) Surfing on a new wave of single-molecule fluorescence methods. Physics and Biology, 7, 031001.[4] Heilemann, M.; van de Linde, S.; Mukherjee, A. & Sauer, M. (2009) Super-resolution imaging with small organic fluorophores. Angewandte Chemie, 48, 6903-6908.