Measuring nanoscale distances by structured illumination microscopy and image cross-correlation spectroscopy (Sim-iccs)

Isotta Cainero, Elena Cerutti, Mario Faretta, Gaetano Ivan Dellino, Pier Giuseppe Pelicci, Alberto Diaspro, Luca Lanzanò

Research output: Contribution to journalArticlepeer-review


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 tech-niques, 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.

Original languageEnglish
Article number2010
Pages (from-to)1-13
Number of pages13
Issue number6
Publication statusPublished - Mar 2 2021


  • Colocalization
  • ICCS
  • Image correlation spectroscopy
  • Image cross-correlation spectroscopy
  • SIM
  • Structured illumination microscopy
  • Super-resolution microscopy

ASJC Scopus subject areas

  • Analytical Chemistry
  • Information Systems
  • Atomic and Molecular Physics, and Optics
  • Biochemistry
  • Instrumentation
  • Electrical and Electronic Engineering


Dive into the research topics of 'Measuring nanoscale distances by structured illumination microscopy and image cross-correlation spectroscopy (Sim-iccs)'. Together they form a unique fingerprint.

Cite this