Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM).
Active stabilization
Biophysics
Drift correction
Localization microscopy
Single-molecule imaging
TIRF microscopy
Journal
Bio-protocol
ISSN: 2331-8325
Titre abrégé: Bio Protoc
Pays: United States
ID NLM: 101635102
Informations de publication
Date de publication:
05 Jul 2021
05 Jul 2021
Historique:
received:
17
12
2020
revised:
16
03
2021
accepted:
05
04
2021
entrez:
30
7
2021
pubmed:
31
7
2021
medline:
31
7
2021
Statut:
epublish
Résumé
The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision. Here, we describe a step-by-step protocol for building a total internal reflection microscope combined with the feedback loops necessary for sample and detection stabilization, which we routinely use in single-molecule localization microscopy (SMLM). The performance of the final microscope with feedback loops, called feedback SMLM, has previously been described. We demonstrate how to build a replica of our system and include a list of the necessary optical components, tips, and an alignment strategy.
Identifiants
pubmed: 34327271
doi: 10.21769/BioProtoc.4074
pii: e4074
pmc: PMC8292130
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e4074Subventions
Organisme : NCI NIH HHS
ID : P30 CA008748
Pays : United States
Informations de copyright
Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.
Déclaration de conflit d'intérêts
Competing interestsThe authors declare no competing financial interests.
Références
Opt Lett. 2020 May 15;45(10):2732-2735
pubmed: 32412453
Nat Methods. 2008 Feb;5(2):159-61
pubmed: 18176568
Science. 2008 Feb 8;319(5864):810-3
pubmed: 18174397
Nat Commun. 2018 Jan 9;9(1):123
pubmed: 29317629
Traffic. 2001 Nov;2(11):764-74
pubmed: 11733042
PLoS One. 2017 Mar 16;12(3):e0173879
pubmed: 28301563
Nat Protoc. 2009;4(11):1623-31
pubmed: 19834477
Sci Adv. 2020 Apr 17;6(16):eaay8271
pubmed: 32494604
Nat Protoc. 2021 Jan;16(1):497-515
pubmed: 33268882
Biomed Opt Express. 2015 Jan 06;6(2):277-96
pubmed: 25780724
Cell. 2015 Dec 3;163(6):1314-25
pubmed: 26638068
Curr Protoc Cytom. 2009 Oct;Chapter 12:Unit12.18
pubmed: 19816922
Nat Protoc. 2014 May;9(5):1083-101
pubmed: 24722406
Sci Rep. 2018 Sep 17;8(1):13912
pubmed: 30224660
Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3125-30
pubmed: 19202073
Nat Methods. 2008 May;5(5):417-23
pubmed: 18408726
Opt Lett. 2014 Oct 15;39(20):6013-6
pubmed: 25361143
Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):2995-9
pubmed: 19211795
J Am Chem Soc. 2020 Apr 1;142(13):6251-6260
pubmed: 32129999
Phys Rev Lett. 2014 Sep 26;113(13):133902
pubmed: 25302889
Nat Methods. 2014 Mar;11(3):313-8
pubmed: 24487583
Cell. 2016 Aug 11;166(4):1028-1040
pubmed: 27397506
Nature. 2009 Feb 26;457(7233):1159-62
pubmed: 19098897