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Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)

Authors :
Isotta Cainero
Elena Cerutti
Mario Faretta
Gaetano Ivan Dellino
Pier Giuseppe Pelicci
Alberto Diaspro
Luca Lanzanò
Source :
Sensors, Vol 21, Iss 6, p 2010 (2021)
Publication Year :
2021
Publisher :
MDPI AG, 2021.

Abstract

Since the introduction of super-resolution microscopy, there has been growing interest in quantifying the nanoscale spatial distributions of fluorescent probes to better understand cellular processes and their interactions. One way to check if distributions are correlated or not is to perform colocalization analysis of multi-color acquisitions. Among all the possible methods available to study and quantify the colocalization between multicolor images, there is image cross-correlation spectroscopy (ICCS). The main advantage of ICCS, in comparison with other co-localization techniques, is that it does not require pre-segmentation of the sample into single objects. Here we show that the combination of structured illumination microscopy (SIM) with ICCS (SIM-ICCS) is a simple approach to quantify colocalization and measure nanoscale distances from multi-color SIM images. We validate the SIM-ICCS analysis on SIM images of optical nanorulers, DNA-origami-based model samples containing fluorophores of different colors at a distance of 80 nm. The SIM-ICCS analysis is compared with an object-based analysis performed on the same samples. Finally, we show that SIM-ICCS can be used to quantify the nanoscale spatial distribution of functional nuclear sites in fixed cells.

Details

Language :
English
ISSN :
14248220
Volume :
21
Issue :
6
Database :
Directory of Open Access Journals
Journal :
Sensors
Publication Type :
Academic Journal
Accession number :
edsdoj.83a6ffc3f8ec45fb8ff1dd4054e7817e
Document Type :
article
Full Text :
https://doi.org/10.3390/s21062010