We develop novel optical methods and instrumentation for the observation of biological phenomena with the highest fidelity. In particular, we focus on super resolution fluorescence imaging and single molecule tracking implementations that offer outstanding spatial and/or temporal resolution. By bringing together expertise in optics, electronics, data processing, labelling and photophysics, we develop solutions to transversal obstacles in biology and biophysics such as quantification of protein clusters and complexes, and identification of protein interaction and dynamics.
Super resolution microscopy
Fluorescence microscopy is an invaluable tool for exploring the structure and function of biological processes. It provides high specificity and contrast for the observation of cellular components (DNA, RNA, proteins, lipids, etc.) tagged with fluorescent molecules in a minimally invasive fashion, even allowing the study of live specimens. The spatial resolution of classical fluorescence microscopy is limited tohundreds of nanometers due to the diffraction of light; however, higher resolutions were unlocked with the development of the so-called super-resolution methodologies (stimulated emission depletion (STED) microscopy, photo-activated localization microscopy/stochastic optical reconstruction microscopy (PALM/STORM), among others)
In the last decade, achieving resolutions in the order of 10 nm to 100 nm became routine and has revealed details of subcellular organelles and new structures as well as ultrastructural anatomy in tissue, granting the Nobel Prize in Chemistry in 2014 to the developers of super resolution. Despite this revolution, the development of an ultimate microscope –revealing the precise nanometric location of all molecules of interest at all times without affecting the sample under study– remains elusive. Among several adversities, the photon budget of fluorescent probes is a fundamental bound for the trade-off between spatial and time resolution.
Advanced light microscopy and biophysics
How can light-based methods provide maximal information on biological processes? What are the limits to the information we can extract? How deep and fast can we look? How can we use extreme resolution to answer concrete questions? What answers can multimodal approaches deliver?
The development of novel optical methods like MINFLUX (see below) shows that the answers to these questions remain unexplored or incomplete, and that there is much to gain from synergistically combining expertise from diverse areas like optics, electronics, statistics, chemistry and biology. By solving transverse methodological challenges, we strive to push forward the maturation of light microscopy and profoundly influence life sciences along the way.
To overcome this, maximally informative luminescence excitation or MINFLUX, merges elements of information theory with (i) the single-emitter nature of PALM/STORM (fig. 1A) and (ii) the beam geometries typically used in STED (e.g the so-called “doughnut” beam). This technique (fig. 1 B–E) has shown that the information that each photon contains on the location of its emitter is a flexible quantity and that it can be dramatically increased in imaging and tracking applications (fig. 2B–D). Therefore, a given localization precision can be obtained by using much fewer photons (e.g. 20 times) than in conventional centroid-localization techniques, such as PALM and STORM.
Figure 1. (A) Principle of PALM/STORM, where the resolution depends on the wavelength, numerical aperture and number of collected photons. (B) MINFLUX depicted as a ruler, where emitters are located from a sequence of exposures to tailored light patterns. The resolution now depends on the size of the ruler (L, separation between sequential excitations), instead of the wavelength. (C) A MINFLUX scheme in 2D with multiple excitations with doughnut-shaped beams, each coloured dot is the center of an exposure. (D) A MINFLUX scheme in 3D, with a beam created by top-hat wavefront shaping. (E) Iterative MINFLUX, where successive approximations to the location of the molecule produce a localization that surpasses the typical N-1/2 dependence..
Figure 2. (A) MINFLUX nanoscopy by sequentially localizing individually blinking fluorophores; a DNA-origami arrangement is imaged. (B) MINFLUX tracking of a DNA-origami flipping device, a fluorophore is followed with 2.5 nm precision every ~0.5 ms. (C) MINFLUX tracking of the small ribosomal subunit protein in living E. coli. (D) Iterative MINFLUX imaging of nuclear pore complex in 3D (fixed) and 2D (live, with comparison to classical SMLM), and of the PSD-95 protein at a neuronal synapse.
Senior Research Assistant
- Since 2020 Research Group Leader at IMP, Vienna, Austria
- 2012–2019 Post Doctoral Research at MPI for Biophysical Chemistry, Göttingen, Germany
- 2007–2012 PhD at University of Buenos Aires, Argentina
- 2002–2007 Electrical Engineering at University of Buenos Aires, Argentina
- 2020–2025 Starting Grant from the European Research Council.
- >30 Invitations to lectures, seminars and international conferences.
Positions calls are now open (last update 10.02.2020).
Find us at the following events:
Research Institute for Molecular Pathology
1030, Vienna, Austria