Cargando…

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

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 coloca...

Descripción completa

Detalles Bibliográficos
Autores principales: Cainero, Isotta, Cerutti, Elena, Faretta, Mario, Dellino, Gaetano Ivan, Pelicci, Pier Giuseppe, Diaspro, Alberto, Lanzanò, Luca
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001887/
https://www.ncbi.nlm.nih.gov/pubmed/33809144
http://dx.doi.org/10.3390/s21062010
_version_ 1783671334874644480
author Cainero, Isotta
Cerutti, Elena
Faretta, Mario
Dellino, Gaetano Ivan
Pelicci, Pier Giuseppe
Diaspro, Alberto
Lanzanò, Luca
author_facet Cainero, Isotta
Cerutti, Elena
Faretta, Mario
Dellino, Gaetano Ivan
Pelicci, Pier Giuseppe
Diaspro, Alberto
Lanzanò, Luca
author_sort Cainero, Isotta
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8001887
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-80018872021-03-28 Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS) Cainero, Isotta Cerutti, Elena Faretta, Mario Dellino, Gaetano Ivan Pelicci, Pier Giuseppe Diaspro, Alberto Lanzanò, Luca Sensors (Basel) Article 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. MDPI 2021-03-12 /pmc/articles/PMC8001887/ /pubmed/33809144 http://dx.doi.org/10.3390/s21062010 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Cainero, Isotta
Cerutti, Elena
Faretta, Mario
Dellino, Gaetano Ivan
Pelicci, Pier Giuseppe
Diaspro, Alberto
Lanzanò, Luca
Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)
title Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)
title_full Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)
title_fullStr Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)
title_full_unstemmed Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)
title_short Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)
title_sort measuring nanoscale distances by structured illumination microscopy and image cross-correlation spectroscopy (sim-iccs)
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001887/
https://www.ncbi.nlm.nih.gov/pubmed/33809144
http://dx.doi.org/10.3390/s21062010
work_keys_str_mv AT caineroisotta measuringnanoscaledistancesbystructuredilluminationmicroscopyandimagecrosscorrelationspectroscopysimiccs
AT ceruttielena measuringnanoscaledistancesbystructuredilluminationmicroscopyandimagecrosscorrelationspectroscopysimiccs
AT farettamario measuringnanoscaledistancesbystructuredilluminationmicroscopyandimagecrosscorrelationspectroscopysimiccs
AT dellinogaetanoivan measuringnanoscaledistancesbystructuredilluminationmicroscopyandimagecrosscorrelationspectroscopysimiccs
AT peliccipiergiuseppe measuringnanoscaledistancesbystructuredilluminationmicroscopyandimagecrosscorrelationspectroscopysimiccs
AT diasproalberto measuringnanoscaledistancesbystructuredilluminationmicroscopyandimagecrosscorrelationspectroscopysimiccs
AT lanzanoluca measuringnanoscaledistancesbystructuredilluminationmicroscopyandimagecrosscorrelationspectroscopysimiccs