Establishing super-resolution microscopy in the Barcelona region
Dr. Timo Zimmermann, Advanced Light Microscopy Unit, Centre for Genomic Regulation, and Dr. Pablo Loza-Alvarez, Super Resolution Light Microscopy and Nanoscopy lab at ICFO, Barcelona, Spain
The talk presents a short overview of the past and current activities in the two institutes hosting this meeting, ICFO and CRG in the field of Super-Resolution (SR) microscopy. Both institutes have established a range of SR instruments and in addition to scientific cooperations the microscopy units are working together since 2010 in the Super-Resolution Light Nanoscopy Alliance (SLN@BCN). In addition tointe rnal users they are aiming to provide access to SR technologies at the European and national level and are participating in various aspects of the Euro-BioImaging initiative. The talk will cover the different applications of the several available SR instruments and the challenges faced in following a fast developing field and adapting the user access models accordingly.
Combining STED and array tomography microscopy
Dr. Alberto Lleó, Neurology Department, Hospital de Sant Pau, Barcelona, Spain
Array Tomography microscopy (ATM) is a Super-Resolution technique specially suited for the study of synapses. ATM overcomes the z-resolution limitation of conventional confocal by ultrathin sectioning of samples into ribbons of 70 nm followed by immunofluorescence imaging and three-dimensional reconstructions of structures of interest. ATM allows precise quantification of the number, volume, and protein labelling of small structures in a high-throughput semiautomated manner. In our group we have applied ATM to the study of human brain tissue to observe synaptic changes in different neurodegenerative conditions, such us Alzheimer’s disease (AD) and dementia with Lewy bodies. In AD, the accumulation of amyloid plaques in the extracellular space of the brain is one of the main hallmarks of the disease. Amyloid plaques are composed of the aggregation of the amyloid-β (Aβ) peptide. It is not yet well established which Aβ aggregation state prevails in vivo although Aβ oligomers have suggested to be particularly toxic for synapses. However, the small size of Aβ oligomers makes traditional imaging techniques not adequate. Here we present the combination of STED and Array Tomography Microscopy (ATM) to image Aβ oligomers in human brain obtained from two patients with AD. In collaboration with the ICFO (Barcelona) we show that oligomeric assemblies can be differentiated from mature fibrils in human brain with nanoscale resolution.
- Micheva KD, et al. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron. 2007 Jul 5;55(1):25-36.
- Kay KR, et al. Studying synapses in human brain with array tomography and electron microscopy. Nat Protoc. 2013;8(7):1366-80.
- Pickett EK, et al. Non-Fibrillar Oligomeric Amyloid-β within Synapses. J Alzheimers Dis. 2016 May ;53(3):787-800.
Silicon-rhodamines - far-red fluorogenic fluorophore for the imaging of cellular structures
Dr. Gražvydas Lukinavicius, Max-Planck-Institute for Biophysical Chemistry, Department NanoBiophotonics, Göttingen, Germany
The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths, fluorogenic and can be flexibly implemented in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proven to be extremely difficult. This shortcoming was addressed by examining several candidates belonging to the rhodamine class which have different substitutes at position 10 of the xanthene dye: tetramethylrhodamine (oxygen), carbopyronine (carbon) and silicon-rhodamine (silicon). Silicon-rhodamines (SiR) display superior fluorogenic properties compared to the regular tetramethylrhodamine or carbopyronine. The reason is identified in their spirolactonization innate sensitivity for the dielectric constant of the microenvironment. As a result, SiR probes targeting actin, tubulin, DNA and lysosomes were developed and successfully employed in imaging these structures in living cells using STED and SIM microscopies. Combination of SiR and SIR700, or SiR and carbopyronine enables multicolor imaging and positioning of several cellular structures. Furthermore, it suggest that the SIRs are potentially useful fluorophores for the generation of the fluorogenic probes targeting different structures in the cell.
- A near-infrared fluorophore for live-cell Super-Resolution microscopy of cellular proteins. Nat Chem 2013, 5, 132-139.
- Fluorogenic probes for live-cell imaging of the cytoskeleton. Nature Methods 2014, 11(7), 731-733.
- SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat Commun 2015, 1;6:8497.
- Fluorogenic probes for multicolor imaging in living cells. J Am Chem Soc. 2016, 138 (30), pp 9365–9368.
DNA in new roles: quantifying super-resolution and increasing photon yields
The number of photons per molecule and their brightness ultimately determine the resolution and signal-to-noise ratio in Super-Resolution microscopy and single-molecule detection. We will present the development of Super-Resolution microscopy from the viewpoint of the fluorescent probes used and discuss the role of photostability . We will then demonstrate recent advances to use the full photon budget from single dye molecules in the Super-Resolution technique DNA PAINT [3-5]. DNA PAINT is especially powerful in combination with DNA nanotechnology and has yielded the highest resolution with DNA origami nanorulers. Such nanorulers have become the prime reference to demonstrate resolution and as positive controls for Super-Resolution microscopes . We also show how imaging next to gold nanoparticles is distorted by plasmonic effects yielding a single-molecule mirage. Using self-assembled DNA origami structures we quantify the plasmonic influences that also include increased brightness [7, 8] and photostability  of fluorescent dyes.
- Hell, S.H. et al. The 2015 Super-Resolution microscopy roadmap. Journal of Physics D: Applied Physics 48, 443001 (2015).
- Vogelsang, J. et al. Make them Blink: Probes for Super-Resolution Microscopy. Chemphyschem 11, 2475-2490 (2010).
- Molle, J. et al. Super-Resolution microscopy with transient binding. Curr Opin Biotechnol 39, 8-16 (2016).
- Raab, M., Schmied, J.J., Jusuk, I., Forthmann, C. & Tinnefeld, P. Fluorescence microscopy with 6 nm resolution on DNA origami. Chemphyschem 15, 2431-2435 (2014).
- Jungmann, R. et al. Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami. Nano Lett 10, 4756-4761 (2010).
- Schmied, J.J. et al. DNA origami-based standards for quantitative fluorescence microscopy. Nat Protoc 9, 1367-1391 (2014).
- Puchkova, A. et al. DNA Origami Nanoantennas with over 5000-fold Fluorescence Enhancement and Single-Molecule Detection at 25 muM. Nano Lett 15, 8354-8359 (2015).
- Acuna, G.P. et al. Fluorescence enhancement at docking sites of DNA-directed self-assembled nanoantennas. Science 338, 506-510 (2012).
- Pellegrotti, J.V. et al. Controlled reduction of photobleaching in DNA origami-gold nanoparticle hybrids. Nano Lett 14, 2831-2836 (2014).
Super-resolution microscopy of HIV-1 assembly and maturation
Prof. Dr. Hans-Georg Kräusslich, Department of Infectious Diseases, Virology, Hospital University Heidelberg, Germany
Human immunodeficiency virus particles assemble at the plasma membrane of the infected cell, where the structural viral Gag polyproteins, the viral envelope (Env) glycoproteins, the viral genome and several other viral and cellular factors gather to form the nascent virion. Following release from the cell, the initial immature virion undergoes maturation to the infectious virus. Morphological maturation is triggered by cleavage of the structural polyproteins Gag and GagPol by the virus encoded protease (PR). The ensuing complete remodeling of the virion architecture is essential for HIV infectivity. This maturation process appears to be highly regulated, but dynamic information is currently lacking.
We have analyzed HIV-1 assembly sites and extracellular immature and mature particles using various Super-Resolution microscopy and have described the distribution of viral and cellular proteins and their dynamic changes. Using HIV-1 derivatives carrying fluorescent labels within Gag or GagPol we employed STED and 4Pi STORM nanoscopy to visualize virion substructures and study the dynamics of HIV-1 polyprotein processing in situ. The obtained Super-Resolution images confirmed the incompleteness of the immature Gag shell based on electron tomography of frozen samples, revealed novel information about the arrangement of GagPol in the immature virion and allowed clear visual distinction between the immature and mature particle phenotype. Employing a recently described photolabile PR inhibitor we were able to trigger Gag processing within purified HIV-1 particles under nanoscopic observation and followed the maturation process over time. This approach allowed us to determine the half-life of ~30 minutes for HIV-1 Gag processing in situ. This work provides a basis for future studies of HIV-1 maturation in nascent virions at the host cell plasma membrane.
Comparison of DNA labels for STED microscopy
Dr. Steffen Dietzel, Biomedical Center (BMC), Ludwig-Maximilians-Universität München (LMU) and Walter-Brendel-Zentrum für Experimentelle Medizin, LMU, Munich, Germany
Stimulated emission depletion (STED) microscopy is one of the more widely applied Super-Resolution techniques. It combines the advantages of breaking the diffraction barrier with comparatively fast image generation and relative ease of use in commercial systems. In our research environment, many groups are interested in the architecture of chromatin. Unfortunately, when we started this study no DNA specific dye was described to work well with STED in mammalian cells. We therefore investigated a number of DNA dyes and labels for their suitability. A good STED dye is bright and can be efficiently depleted by the STED laser. Importantly, the dye should not be excitable by the depletion laser.
After a preselection of nucleic acid stains that mostly stain the cell nucleus (and less the cytoplasm and thus RNA) five DNA-binding fluorochromes were selected for further investigation, DAPI, Hoechst, SYTO 16, SYTO 21 and SIR-DNA. In addition to these, we investigated DNA replication labelling with EdU and detected with Alexa 488, 555 and 594. After long enough incubation, dividing cells will have their complete chromatin labeled by this technique. Alexa 594 was previously described as a good fluorochrome for STED and thus was expected to provide a positive control.
On our Leica SP8 STED 3X, each label was imaged first in confocal mode and then imaged with increasing powers of the depletion laser. Depletion at 592, 660, and 775 nm was used if possible with the respective dye. In addition, the intensity of the anti-Stokes excitation by the respective depletion laser was measured for each dye. Due to the large size of the depletion PSF, anti-Stokes excitation, if present, has the potential of substantially decreasing the achieved resolution. For increasing depletion laser intensities, our data show that many dyes have a sweet spot, where depletion is already relatively high, but anti-Stokes excitation yet low. The best results in terms of good depletion and low anti-Stokes excitation were indeed obtained with EdU-Alexa 594 label, if the labelling was performed at the right concentration. Furthermore, relatively low bleaching also allowed 3D recordings of entire nuclei with Super-Resolution. More detailed results will be presented at the meeting.
Resolving signalling platforms for cell growth
Dr. Valeria Caiolfa, Microscopy and Dynamic Imaging Unit, CNIC, Madrid
We are involved in a collaborative project that explores cell membrane domains functioning as signalling platforms for cell growth. By confocal imaging the domains appear as sharply-defined areas of coalescence of several signalling molecules including Trops, tetraspanins and GF-receptors. They measure tens of microns and are dynamically recurrent and with unexpected stability over time. We are exploring the organization and heterogeneity of TROP and tetraspanin molecules inside and outside the domains by STED Super-Resolution microscopy. (In collaboration with Saverio Alberti, University of Chieti, Chieti, Italy)
Super-Resolution Localization Microscopy: A chemical viewpoint
Dr. Lorenzo Albertazzi, Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain
Super-resolution imaging techniques based on single molecule localization such as STORM, PALM, GSD, PAINT and many other variants showed a tremendous potential thanks to their nanometric resolution, 3D and multicolor abilities. As the density and the photochemical control of individual dye molecules are key for these techniques is not surprising that the chemical aspects of the methods are of crucial importance to achieve a high quality imaging. In this talk I present a chemical viewpoint on super resolution microscopy discussing issues such as labelling strategy, choice of the dyes, photoswithing mechanisms and buffer preparation and how to choose and optimize them to obtain the best performances. Recent advances on dye synthesis, labelling procedures and buffer preparation will be highlighted. Novel applications of Super-Resolution imaging in fields such as materials chemistry will be presented, demonstrating that the potential of super resolution exceed the use in cell and molecular biology. In particular developments of novel STORM and PAINT methods to study the structure and the dynamics of bio-inspired materials in vitro and their interactions with cells will be showed.