Techniques and methods

Imaging

Laser scanning microscopes, fluorescent lifetime imaging (FLIM) with phasor analysis (University of Jyväskylä)

Spinning disk microscope (SDM), Centre for Structural Systems Biology (CSS), Hamburg, GER) (Prof. Jens Bosse)

Structured illumination microscope (SIM), Tampere Imaging Facility (Dr Teemu Ihalainen

Expansion microscopy

In the ExM technique, the proteins of interest (e.g., viral capsids, proteins, cellular proteins) are labeled with fluorescent antibodies, treated with proteinase, and linked to a swelling polymer. The physical expansion of the polymer enables super-resolution microscopy with LSCM.

Publication

Mäntylä E, Montonen T, Azzari L, Mattola S, Hannula M, Vihinen-Ranta M, Hyttinen J, Vippola M, Foi A, Nymark S, Ihalainen TO (2023).^.Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina. Mol Biol Cell, 34(9):br13.

Cryo soft X-ray tomography

Performed in:

National Center for X-ray Tomography, Lawrence Berkeley National Laboratory (Prof. Carolyn Larabell, Berkeley, CA)

ALBA Synchrotron Light Source, Barcelona (Dr. Eva Pereiro)

Sirius XT (Dublin, Dr Kenneth  Fahy)

Volume EM

Focused ion-beam scanning electron microscopy (FIB-SEM),

Serial block face scanning electron microscopy (SBF-SEM)

University of Helsinki (Eija Jokitalo)

Protein-protein interactions

Proximity ligation assay (PLA) analysis

BioID (In collaboration with the Proteomics Unit, University of Helsinki, Institute of Biotechnology with Dr. Kari Salokas, Research Director Markku Varjosalo)

rRNA Molecular Biology

Laboratory of RNA Molecular Biology, Université libre de Bruxelles (Prof. Denis LJ Lafontaine)

Data analyses

The fluorescence imaging data is analyzed by in-house ImageJ analysis pipelines and machine learning. e.g., viral capsids have been tracked using the TrackMate plugin in Image J software and analyzed using a code to correlate mobility to local chromatin density. FLIM and FLIM-FRET data will be analyzed by decay fitting and phasor analysis tool in LA SX (Leica), and the rRNA processing will be done by the JACUSA2 software (50). EM, FIB-SEM, and SXT data will be segmented and analyzed with ImageJ, Amira, Microscopy Image Browser (MIB), eC-CLEM, and SXT algorithms produced at Lawrence Berkeley National Laboratory (CA).