Digitala Vetenskapliga Arkivet

Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes
Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
Show others and affiliations
2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 355, no 6325, p. 606-612Article in journal (Refereed) Published
Abstract [en]

We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolutionmicroscopy, MINFLUXattained similar to 1-nanometer precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

Place, publisher, year, edition, pages
2017. Vol. 355, no 6325, p. 606-612
National Category
Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-317586DOI: 10.1126/science.aak9913ISI: 000393636700043PubMedID: 28008086OAI: oai:DiVA.org:uu-317586DiVA, id: diva2:1083812
Funder
EU, European Research CouncilKnut and Alice Wallenberg FoundationAvailable from: 2017-03-22 Created: 2017-03-22 Last updated: 2020-05-07Bibliographically approved
In thesis
1. Bacterial DNA repair and molecular search
Open this publication in new window or tab >>Bacterial DNA repair and molecular search
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Surveillance and repair of DNA damage is necessary in all kinds of life. Different types of DNA damage require different repair mechanisms, but these mechanisms are often similar in all domains of life. The most serious type of damage, double stranded DNA breaks, are for example repaired in conceptually similar ways in both bacteria and eukaryotes. When this kind of breaks are repaired by homologous recombination, a homology to the site of the break must be found. Sometimes, this homology can be located far away from the break necessitating a search. Considering the large amount of heterologous DNA present, the complexity of this search is enormous. If and how this search can proceed has been unclear even in simple and well characterized organisms as E. coli.

In this thesis, microscopy together with microfluidics are used to show that DNA repair by homologous recombination occurs even between homologies separated by several micrometers. We also see that it finishes well within the time of a cell generation, with the enigmatic search phase being as quick as eight or possibly even three minutes. Since this time is much faster than expected, we present a physical model demonstrating how homology search on this time scale is indeed plausible. Based on these results, we conclude that homologous repair using distantly located templates is likely to be a physiologically relevant mechanism of DNA repair.

Microscopy together with image analysis by deep learning also provides a new method of detecting DNA damage in real time. Combined with tracking of cell lineages, it reveals that DNA damage in E. coli is repaired efficiently enough that the resulting fitness cost is close to none. With the same methods we also study the effect of deletions of several DNA repair enzymes, and largely confirms their previous characterizations. Among these, we confirm that the intriguing RecN protein is important but not absolutely necessary in DSB repair, that it acts early, and possibly aids in physically shaping the structure mediating the search.

In addition to this, it is shown how DNA transcription and translation modulates the shape of the E. coli nucleoid. We observe how strong a transcription of a gene within a few minutes moves the gene towards the periphery of the cell where the concentration of ribosomes is higher, a movement possibly also aided by protein translation.

We also present MINFLUX, a microscope for both nanometer scale localization of single fluorophores as well as in vivo single particle tracking with unprecedented trace length and resolution. Using this, the E. coli small ribosomal subunit could be observed to quickly shift between fast and slow diffusion states which might represent probing and discarding of RNAs suitable for translation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. p. 77
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1947
Keywords
DNA repair, DNA damage, homologous recombination, homologous repair, recombination, RecA, I-SceI
National Category
Bioinformatics and Systems Biology Microbiology
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-410039 (URN)978-91-513-0966-8 (ISBN)
Public defence
2020-08-28, A1:111a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2020-06-04 Created: 2020-05-07 Last updated: 2020-06-04

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Gynnå, Arvid H.Elf, Johan
By organisation
Science for Life Laboratory, SciLifeLabMolecular Systems Biology
In the same journal
Science
Biophysics

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 946 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf