"People really interact during the sessions – this is really important and helps to create a nice atmosphere. I look forward to participating in the 3rd meeting", said Giuseppe Vicidomini, one of the speakers at this year's meeting, who specializes in gated confocal super-resolution techniques at the IIT, Genoa, Italy.
"The meeting enables users of Leica Microsystems ground-breaking super-resolution systems to meet and share experiences – we are happy to provide a platform for our customers to do that, and to continue to support the work they do in research science" says Baba Awopetu, European Marketing Director, Leica Microsystems.
Super-resolution at the NKI: Where do we come from, where are we going?
Kees Jalink, The Netherlands Cancer Institute, Amsterdam, The Netherlands
For centuries, the light microscope has played a pivotal role in biology and medicine. Without the ability to observe cells and tissues, to study their morphology, dynamics and molecular composition, we would have only a fraction of the understanding of life we have today. Yet, until recently, we failed to have the resolution to observe the building blocks of life, as the resolution of light microscopy is about 50-fold too low. Super-resolution microscopy is rapidly changing that. We use Ground State Depletion Imaging to study the composition of the cytoskeleton and to decipher signal transduction mechanisms.
As a first step, we have invested in optimizing preparation and recording conditions. Blinking conditions have been optimized and algorithms were developed for drift correction and background subtraction. We are currently applying SR-GSD in a number of biological studies. In collaboration with the Sonnenberg lab in our institute, we have focussed on the structure of hemidesmosomes, adhesive structures within the skin. We also study keratin filament formation and plasticity in collaboration with Dr. Reinhard Windhoffer in Aachen, Germany. In my talk, I will update you on developments in our lab and focus on what will be – I think – the important directions for the near future of localization microscopy.
Encoding and decoding spatio-temporal information for super-resolution microscopy
Luca Lanzanó, Italian Institute of Technology, Genoa, Italy
The visualization at the nanoscale level inside cells has become a fundamental need in molecular biology. Most of the super-resolution fluorescence methods developed so far provide sub-diffraction resolution by transiently switching the fluorophores between optically distinguishable states. Accordingly, the different super-resolution concepts have been broadly classified into stochastic and targeted switching methods, based on how the fluorophore states are manipulated . However, sub-diffraction resolution can also be obtained with a very different strategy: by encoding spatial information into a temporal channel and decoding it after the transmission through the microscope setup . The nanoscale spatial distribution of the molecules inside the detection volume of the microscope can be encoded within the fluorescence dynamics and can be decoded by resolving the signal into its dynamics components. Thus the challenge of increasing the spatial resolution of an optical microscope is translated into the spectroscopy task of resolving the signal into its dynamics components. We present here a robust and general method to spatially sort the fluorescence photons on the basis of the associated molecular dynamics . In this method, the separation of dynamics components is obtained without any fitting procedure using phasor analysis . In a specific implementation, we consider the gradients in fluorescence lifetime generated by stimulated emission in a CW-STED microscope . We show that spatial resolution can be increased indefinitely by increasing the number of resolved components up to a maximum, predictable number, determined by the amount of noise. Interestingly the method also isolates any uncorrelated background signal. We demonstrate that this spectroscopy-based approach provides background-free nanoscale imaging of subcellular structures, opening new routes in super-resolution microscopy based on the encoding of spatial information through manipulation of molecular dynamics .
- Hell SW: Far-field optical nanoscopy. Science 316: 1153–58 (2007).
- Enderlein J: Breaking the diffraction limit with dynamic saturation optical microscopy. Applied Physics Letters 87: 094105 (2005).
- Lanzanò L, et al.: Encoding and decoding spatio-temporal information for super-resolution microscopy. Nature Communications 6: 6701 (2015).
- Digman MA, et al.: The phasor approach to fluorescence lifetime imaging analysis. Biophysical Journal 94: L14–16 (2008).
- Vicidomini G, et al.: Sharper low-power STED nanoscopy by time gating. Nature Methods 8: 571–73 (2011).
Deciphering steps of mRNP assembly in developing oocytes using STED microscopy
Imre Gaspar, Developmental Biology Unit, EMBL, Heidelberg, Germany
In vast majority of the studied animal species, body axes are determined already during oogenesis through localization and localized translation of messenger RNA molecules. These mRNAs contain specific structures (cis-acting elements) usually - but not limited to – in their 3’ untranslated region which recruit specific protein molecules (trans-acting elements) resulting in formation of functional ribonucleoprotein complexes (mRNPs) . Composition of mRNPs, however, changes dynamically from translation to decay , making microscopic techniques with high spatial and temporal resolution invaluable for studying mRNP biogenesis.
During Drosophila oogenesis, oskar mRNPs are synthesized in the nurse cells from where they get transported into the interconnected oocyte by cytoplasmic dynein. Within the oocyte, oskar mRNA localizes to the posterior pole initially in a kinesin-1 dependent manner during mid-oogenesis . To understand the formation of transport and localization competent oskar mRNPs, we chose a candidate based co-localization assay using confocal and STED microscopy. To maximize specificity and minimize the distance of label to oskar mRNA, we used forced intercalation of thiazole orange (FIT) probes that only fluoresce when binding to target . FIT probes allow performing "mild" in situ hybridization that preserves auto-fluorescence of GFP tagged proteins. Also, while the emission and excitation spectrum of TO is sufficiently different (red-shifted) from GFP to acquire bleed-through free images, its emission can be stimulated with 592 nm without any re-excitation.
We found that oskar mRNPs associate with kinesin-1 but not with dynein right after export into the nurse cell cytoplasm. Interestingly, association with kinesin-1 requires an atypical tropomyosin protein encoded by the dTropomyosin1 gene and also splicing of the first intron of oskar mRNA .
- Marchand V, Gaspar I, and Ephrussi A: An Intracellular Transmission Control Protocol: assembly and transport of ribonucleoprotein complexes. Current opinion in cell biology 24 (2): 202–10 (2012).
- Gaspar I: Microtubule-based motor-mediated mRNA localization in Drosophila oocytes and embryos. Biochemical Society Transactions 39 (5): 1197–201 (2011).
- Hövelmann F, Gaspar I, Ephrussi A, and Seitz O: Brightness Enhanced DNA FIT-Probes for Wash-free RNA Imaging in Tissue. Journal of the American Chemical Society 135 (50): 19025–32 (2013).
Hövelmann F, Gaspar I, Loibl S, Ermilov EA, Röder B, Wengel J, Ephrussi A, and Seitz O: Brightness through Local Constraint-LNA-Enhanced FIT Hybridization Probes for In Vivo Ribonucleotide Particle Tracking. Angewandte Chemie – International Edition 53 (42): 11370–75 (2014).
- Ghosh S, Marchand V, Gáspár I, and Ephrussi A: Control of RNP motility and localization by a splicing-dependent structure in oskar mRNA. Nature Structural & Molecular Biology 19 (4): 441–9 (2012).
Silicon rhodamine based far-red probes for live cell super-resolution imaging
Luc Reymond, Laboratory of Protein Engineering, École polytechnique fédérale, Lausanne, Switzerland
The ideal fluorescent probe for super-resolution imaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic probes that combine all of these properties has proved to be extremely difficult. Here, we introduce a suite of silicon rhodamine based probes that combine far-red emission, fluorogenicity, minimal cytotoxicity, excellent brightness and photostability for fluorescence imaging of cellular structures in living cells. Applied in stimulated emission depletion (STED) nanoscopy, they allowed imaging of cellular structures in living cells at an unprecedented resolution. The current status of the probes development will be presented together with their applications in live cell imaging.
The architecture of hemidesmosomes as revealed by super resolution microscopy: Quantitative analysis of components
Leila Nahidi Azar, Netherlands Cancer Institute, Amsterdam, The Netherlands
Hemidesmosomes have been extensively studied by immunofluorescence microscopy, but due to its limited resolution, the precise organization of hemidesmosomes remained poorly understood. We studied hemidesmosome organization in cultured keratinocytes by highly corrected 2- and 3-color Super-Resolution microscopy. We observed that in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments. By quantifying molecular distances, we demonstrate that both linker proteins BP230 and plectin interact asymmetrically with keratin. Furthermore, we show that BP180 and BP230 have a characteristic arrangement within these hemidesmosomes with BP180 molecules surrounding a central core of BP230 molecules. In my presentation I will emphasize how novel image analysis methods have to be developed to replace concepts such as colocalization, which are of limited value for SR imaging.
Co-orientation: Quantifying simultaneous co-localization and orientational alignment of filaments in localization microscopy
Robert Nieuwenhuizen, Delft University of Technology, Department of Imaging Physics, Quantitative Imaging Group, Delft, The Netherlands
Cytoskeletal protein networks serve many crucial roles in living cells. To understand how the different filament networks interact and collaborate to perform these functions, it is important to obtain insight into the nanoscale spatial arrangements of these networks. Super-resolution microscopy techniques now make it possible to resolve these networks. This introduces the need for new quantitative tools to interrogate the organization of and mutual interrelations between the different cytoskeletal elements.
We present a new quantitative framework in which we determine how the orientational alignment of filaments affects their colocalization (as measured by the pair cross-correlation function). The simultaneous orientational alignment and colocalization of filaments will be referred to as co-orientation. We show how the strength of the co-orientation can be quantified locally by an anisotropic generalization of Ripley’s K-coefficient and we propose a test for its statistical significance. We demonstrate our methods on simulated localization microscopy data of filament structures, as well as multi-color