Simon Hennig

Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ

Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses.

Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution

We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.

Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

Nanoparticles as Nonfluorescent Analogues of Fluorophores for Optical Nanoscopy

Optical microscopy modalities that achieve spatial resolution beyond the resolution limit have opened up new opportunities in the biomedical sciences to reveal the structure and kinetics of biological processes on the nanoscale. These methods are, however, mostly restricted to fluorescence as contrast mechanism, which limits the ultimate spatial resolution and observation time that can be achieved by photobleaching of the fluorescent probes. Here, we demonstrate that Raman scattering provides a valuable contrast mechanism for optical nanoscopy in the form of super-resolution structured illumination microscopy. We find that nanotags, i.e., gold and silver nanoparticles that are capable of surface-enhanced Raman scattering (SERS), can be imaged with a spatial resolution beyond the diffraction limit in four dimensions alongside and with similar excitation power as fluorescent probes. The highly polarized nature of super-resolution structured illumination microscopy renders these nanotags elliptical in the reconstructed super-resolved images, which enables us to determine their orientation within the sample. The robustness of nanotags against photobleaching allows us to image these particles for unlimited periods of time. We demonstrate this by imaging isolated nanotags in a dense layer of fluorophores, as well as on the surface of and after internalization by osteosarcoma cells, always in the presence of fluorescent probes. Our results show that SERS nanotags have the potential to become highly multiplexed and chemically sensitive optical probes for optical nanoscopy that can replace fluorophores in applications where fluorescence photobleaching is prohibitive for following the evolution of biological processes for extended times.

Quantitative super-resolution microscopy of nanopipette-deposited fluorescent patterns

We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ∼100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3-168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

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.

Survival rate of eukaryotic cells following electrophoretic nanoinjection

Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells.

Label-free Super-resolution Optical Microscopy of Cellular Dynamics

We demonstrate super-resolved structured illumination microscopy (SR-SIM) of Raman-active samples with 100 nm spatial resolution. By combining SR-SIM with coherent Raman scattering, even biological samples can be visualized with doubled spatial resolution.