Three dimensional parallelized RESOLFT nanoscopy for volumetric live cell imaging

Andreas Boden, Francesca Pennacchietti, Ilaria Testa

bioRxiv (Nature Biotechnology accepted, in press) (2020)

The volumetric architecture of organelles and molecules inside cells can only be investigated with microscopes featuring sufficiently high resolving power in all three spatial dimensions. Current methods suffer from severe limitations when applied to live cell imaging such as long recording times and/or photobleaching. By introducing a novel optical scheme to switch reversibly switchable fluorescent molecules, we demonstrate volumetric nanoscopy of living cells with resolution below 100 nm in 3D, large field of view and minimal illumination intensities (W kW/cm2).


Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo

Jes Dreier, Marco Castello, Giovanna Coceano, Rodrigo Cáceres, Julie Plastino, Giuseppe Vicidomini & Ilaria Testa

Nature Communications volume 10, Article number: 556 (2019)

Smart scanning is based on a point scanning RESOLFT setup that avoids unnecessary scanning of parts of the sample not containig structure. This allows for a drastic increase in imaging speed and a smarter use of total illumination dose.


Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

Luciano A. Masullo*, Andreas Bodén*, Francesca Pennacchietti*, Giovanna Coceano, Michael Ratz and Ilaria Testa

Nature Communications volume 9, Article number: 3281 (2018)

Here we present the first RESOLFT based imaging setup able to image living cells in a parrallelized manner with down to 40-60 nm resolution in the lateral plane while maintaining the intrinsic optical sectioning capabilities of a point scanning setup. We demonstrate its performance in several cell types tagged with different reversibly switchable fluorescent proteins.


Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy

Francesca Pennacchietti*, Ekaterina O. Serebrovskaya*, Aline R. Faro*, Irina I. Shemyakina*, Nina G. Bozhanova, Alexey A. Kotlobay, Nadya G. Gurskaya, Andreas Bodén, Jes Dreier, Dmitry M. Chudakov, Konstantin A. Lukyanov, Vladislav V. Verkhusha, Alexander S. Mishin and Ilaria Testa

Nature Methods 15, 601–604 (2018)

Together with our collaborators, we present the reversibly switchable fluorescent proteins (rsFPs) rsFusionRed1-3 and their useability for parallelized RESOLFT imaging using the MoNaLISA imaging platform. The proteins allow for super resolution imaging using only green and red illumination.


Forces drive basement membrane invasion in Caenorhabditis elegans

Rodrigo Cáceres, Nagagireesh Bojanala, Laura C. Kelley, Jes Dreier, John Manzi, Fahima Di Federico, Qiuyi Chi, Thomas Risler, Ilaria Testa, David R. Sherwood, and Julie Plastino

PNAS November 6, 2018 115 (45) 11537-11542


Fluorescent Diarylethene Photoswitches—A Universal Tool for Super‐Resolution Microscopy in Nanostructured Materials

Oleksii Nevskyi, Dmytro Sysoiev, Jes Dreier, Simon Christoph Stein, Alex Oppermann, Florian Lemken, Tobias Janke, Jörg Enderlein, Ilaria Testa, Thomas Huhn, Dominik Wöll (2018)

Small Volume 14, Issue 10


Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy

Katharina N Richter, ..., Giovanna Coceano, ..., Ilaria Testa et al.

The EMBO Journal Volume 37, Issue 1, 2018


Vimentin Levels and Serine 71 Phosphorylation in the Control of Cell-Matrix Adhesions, Migration Speed, and Shape of Transformed Human Fibroblasts

Emmanuel Terriac, Giovanna Coceano, Zahra Mavajian, Tijmen A. G. Hageman, Andreas F. Christ, Ilaria Testa, Franziska Lautenschläger and Annica K. B. Gad

Cells 2017, 6(1), 2


The 2015 Super resolution roadmap

Stefan W. Hell, ..., Ilaria Testa et al.

Journal of Physics D: Applied Physics, 48, 44


CRISPR/Cas9–mediated endogenous protein tagging for RESOLFT super–resolution microscopy of living human cells

Michael Ratz, Ilaria Testa, S.W. Hell and S.Jakobs (2015)

Scientific Reports 5, 9592


Dual Channel RESOLFT Nanoscopy by Using Fluorescent State Kinetics

Ilaria Testa, Elisa D’Este, Nicolai T. Urban, Francisco Balzarotti, Stefan W. Hell (2015)

Nano Letters 15, 103–106

In this paper the concept of RESOLFT super resolution light microscopy was extended to multicolor imaging allowing accurate co-localization studies of multiple proteins simultaneously.


Two–Color RESOLFT Nanoscopy with Green and Red Fluorescent Photochromic Proteins

Flavie Lavoie–Cardinal, Nickels Jensen, Volker Westphal, Andre C. Stiel, Andriy Chmyrov, Jakob Bierwagen, Ilaria Testa, Stefan Jakobs, Stefan W. Hell (2013)

ChemPhysChem doi:10.1002/cphc.201301016


Nanoscopy of living brain slice with low light levels

Ilaria Testa, Nicolai T. Urban, Stefan Jakobs, Christian Eggeling, Katrin I. Willig, Stefan W. Hell (2012)

Neuron 75: 992–1000 doi: 10.1016/j.neuron.2012.07.028

In this paper we showed the potential of RESOLFT super resolution light microscopy for neuroimaging. Neuronal proteins specific for hippocampal dendritic spines were recorded multiple times at high accuracy.


rsEGFP2 enables fast RESOLFT nanoscopy of living cells

Tim Grotjohann*, Ilaria Testa*, Matthias Reuss, Tanja Brakemann, Christian Eggeling, Stefan W Hell, Stefan Jakobs (2012) *equal first author

eLIFE Sciences 1:1–14 doi: 10.7554/eLife.00248

In this paper we pushed the time resolution of RESOLFT super resolution light microscopy. By speeding up the recording time we could follow dynamics of proteins located in various cells compartment.


Diffraction–unlimited all–optical imaging and writing with a photochromic GFP

Tim Grotjohann*, Ilaria Testa*, Marcel Leutenegger*, Hannes Bock, Nicolai T. Urban, Flavie Lavoie Cardinal, Katrin I. Willig, Christian Eggeling, Stefan Jakobs & Stefan W. Hell (2011) *equal first author

Nature 478: 204–208 doi:10.1038/nature10497

In this paper we show, for the first time, the concept of RESOLFT super resolution microscopy to work for biological imaging (30–50nm).


A reversibly photoswitchable GFP–like protein with fluorescence excitation decoupled from switching

Tanja Brakemann, Andre C Stiel, Gert Weber*, Martin Andresen*, Ilaria Testa*, Tim Grotjohann, Marcel Leutenegger, Uwe Plessmann, Henning Urlaub, Christian Eggeling, Markus C Wahl, Stefan W Hell & Stefan Jakobs (2011) *equal first author

Nature Biotechnology 29: 942–950 doi:10.1038/nbt.1952


Nanoscopy of Protein Colocalization in Living Cells by STED and GSDIM

Birka Lalkens, Ilaria Testa, Katrin I. Willig, Stefan W. Hell (2011)

Microscopy Research and Techniques 75:1–6 doi: 10.1002/jemt.21026


Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores, with a Single Wavelength

Ilaria Testa, Christian A. Wurm, Rebecca Medda, Ellen Rothermel, Claas Von Middendorff, Jonas Fölling, Stefan Jakobs, Andreas Schönle, Stefan W. Hell, Christian Eggeling (2010)

Biophysical Journal 99: 2686 – 2694 doi: 10.1016/j.bpj.2010.08.012

In this paper we have shown 3-4 color multiplexed single molecule super resolution imaging.


Understanding biological dynamics: following cells and molecules to track functions and mechanisms

Andrea Palamidessi, Ilaria Testa, Emanuela Frittoli, Sara Barozzi, Massimiliano Garre, Davide Mazza, Pier Paolo Di Fiore, Alberto Diaspro, Giorgio Scita, Mario Faretta (2010)

European Biophysical Journal 39:947-957 doi: 10.1007/s00249-009-0461-x


Far-Field Autofluorescence Nanoscopy

Jakob Bierwagen*, Ilaria Testa*, Jonas Fölling, Dirk Wenzel, Stefan Jakobs, Christian Eggeling, Stefan W. Hell (2010) *equal first author

Nano Letters 10:4249–4252 doi: 10.1021/nl1027638


The Role of the C-Terminus for Functional Heteromerization of the Plant Channel KDC1

Alessia Naso, Ingo Dreyer, Laura Pedemonte, Ilaria Testa, Judith Lucia Gomez-Porras, Cesare Usai, Bernd Mueller-Rueber, Alberto Diaspro, Franco Gambale, Cristiana Picco (2009)

Biophysical Journal 96: 4063-4074 doi: 10.1016/j.bpj.2009.02.0556


Nanoscale separation of molecular species based on their rotational mobility

Ilaria Testa, Andreas Schönle, Claas von Middendorff, Claudia Geisler, Rebecca Medda, Christian A Wurm, Andre C Stiel, Stefan Jakobs, Mariano Bossi, Christian Eggeling, StefanW Hell, and Alexander Egner (2008)

Optic Express 16:21093–21103 doi: 10.1364/OE.16.021093

In this paper we developed a polarization sensitive detection nanoscope based on single molecule super resolution imaging.


Photoactivation of pa–GFP in 3D: optical tools for spatial confinement

Ilaria Testa, Massimiliano Garrè, Dario Parazzoli, Sara Barozzi, Ivan Ponzanelli, Davide Mazza, Mario Faretta, Alberto Diaspro (2008)

European Biophysical Journal, 37: 1219–1227 doi: 10.1007/s00249–008–0317–9


Spatial control of pa–GFP photoactivation in living cells

Ilaria Testa, Dario Parazzoli, Sara Barozzi, Massimiliano Garrè, Mario Faretta, Alberto Diaspro (2008)

Applied Physics Letters 91:13 doi: 10.1063/1.2790847


Blue light (488nm) irradiation induces photo-activation of the photo–activatable green fluorescent protein

Ilaria Testa, Davide Mazza, Sara Barozzi, Mario Faretta, Alberto Diaspro (2007)

Biophysical Journal 96: 4063-4074 doi: 10.1016/j.bpj.2009.02.0556


About SciLifeLab

Our lab is located in the Science for Life Laboratory (SciLifeLab), a national center for molecular biosciences with focus on health and environmental research. SciLifeLab has been created by the coordinated effort of four universities in Stockholm and Uppsala: Stockholm University, Karolinska Institutet, KTH Royal Institute of Technology and Uppsala University.

Open Positions

We are looking for self-motivated and curiosity-driven candidates with an expertise in physics, chemistry or biology, who are looking to make original contributions to the field of super-resolution microscopy. The candidates will work on collaborative projects investigating the nanoscale organization and dynamics of proteins in living neurons and brain tissue with our cutting edge microscopy technology. We offer an outstanding scientific environment and a vibrant working climate with individual freedom and various possibilities for professional development. The work will be funded by the European Union within the ERC project “MoNaLISA”.

Get in Touch

    Ilaria Testa, PhD.
    Associate Professor
    KTH Royal Institute of Technology
    Dept. of Applied Physics
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  • Address:
    Science for Life Laboratory
    Tomtebodavägen 23A
    171 65 Stockholm

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