Lifetime-based super-resolution and its application to a model of oncogenesis
Luca Lanzanò 1,2, Elena Cerutti1,2, Anna P. Privitera 1, Elisabetta Di Franco 1, Morgana D’Amico 1, Pier Giuseppe Pelicci 3,4, Mario Faretta 3, Gaetano Ivan Dellino 3,4, Alberto Diaspro 2,5
1- Department of Physics and Astronomy “Ettore Majorana”, University of Catania, Catania, Italy. 2- Nanoscopy, CHT Erzelli, Istituto Italiano di Tecnologia, Genoa, Italy. 3- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, 20100 Milan, Italy. 4- Department of Oncology and Hemato-Oncology, University of Milan, 20100 Milan, Italy. 5- DIFILAB, Department of Physics, University of Genoa, Genoa, Italy
The development of superresolution microscopy has opened the way for studying biological processes at the nanoscale. I will discuss how to take advantage of fluorescence lifetime detection and phasor analysis to make superresolution microscopy more efficient. In particular, I will discuss the separation of photons by lifetime tuning (SPLIT) method and its implementation in different types of imaging modalities. Then, I will discuss application of superresolution microscopy to the investigation of chromatin alterations in a model of oncogene activation. We apply superresolution microscopy in combination with image cross correlation spectroscopy (ICCS) to an in vitro model of Acute Promyelocytic Leukemia (APL) that allows us to activate the expression of the PML-RARα oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes. In particular, superresolution is combined with a robust image analysis protocol that quickly produces a value of colocalization fraction on several hundreds of single cells and allows observation of cell-to-cell variability.