<ddi:DDIInstance xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="ddi:instance:3_3 http://ddialliance.org/Specification/DDI-Lifecycle/3.3/XMLSchema/instance.xsd" xmlns:xsd="http://www.w3.org/2001/XMLSchema" xmlns:ddi="ddi:instance:3_3" xmlns:r="ddi:reusable:3_3" xmlns:s="ddi:studyunit:3_3" xmlns:d="ddi:datacollection:3_3" xmlns:a="ddi:archive:3_3" xmlns:c="ddi:conceptualcomponent:3_3" xmlns:cm="ddi:comparative:3_3" xmlns:g="ddi:group:3_3" xmlns:l="ddi:logicalproduct:3_3" xmlns:p="ddi:physicaldataproduct:3_3" xmlns:pi="ddi:physicalinstance:3_3" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xhtml="http://www.w3.org/1999/xhtml" xmlns:xml="http://www.w3.org/XML/1998/namespace" isMaintainable="true" scopeOfUniqueness="Agency">
  <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451:0</r:URN>
  <r:Agency>SND</r:Agency>
  <r:ID>doi-10-5281-zenodo-8006451</r:ID>
  <r:Version>0</r:Version>
  <g:ResourcePackage>
    <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.ResourcePackage:2.0</r:URN>
    <r:OtherMaterialScheme>
      <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.OtherMaterialScheme:2.0</r:URN>
    </r:OtherMaterialScheme>
    <a:OrganizationScheme>
      <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.OrganizationScheme-0:2.0</r:URN>
      <a:Individual>
        <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.Individual-0:2.0</r:URN>
        <r:UserAttributePair>
          <r:AttributeKey>affiliation</r:AttributeKey>
          <r:AttributeValue>Demirbay</r:AttributeValue>
        </r:UserAttributePair>
        <a:IndividualIdentification>
          <a:IndividualName>
            <a:LastFamily>Baris</a:LastFamily>
            <a:FullName>
              <r:String>Baris</r:String>
            </a:FullName>
          </a:IndividualName>
        </a:IndividualIdentification>
      </a:Individual>
      <a:Individual>
        <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.Individual-0:2.0</r:URN>
        <r:UserAttributePair>
          <r:AttributeKey>affiliation</r:AttributeKey>
          <r:AttributeValue>Sandberg</r:AttributeValue>
        </r:UserAttributePair>
        <a:IndividualIdentification>
          <a:IndividualName>
            <a:LastFamily>Elin</a:LastFamily>
            <a:FullName>
              <r:String>Elin</r:String>
            </a:FullName>
          </a:IndividualName>
        </a:IndividualIdentification>
      </a:Individual>
      <a:Individual>
        <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.Individual-0:2.0</r:URN>
        <r:UserAttributePair>
          <r:AttributeKey>affiliation</r:AttributeKey>
          <r:AttributeValue>Piguet</r:AttributeValue>
        </r:UserAttributePair>
        <a:IndividualIdentification>
          <a:IndividualName>
            <a:LastFamily>Joachim</a:LastFamily>
            <a:FullName>
              <r:String>Joachim</r:String>
            </a:FullName>
          </a:IndividualName>
        </a:IndividualIdentification>
      </a:Individual>
      <a:Individual>
        <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.Individual-0:2.0</r:URN>
        <r:UserAttributePair>
          <r:AttributeKey>affiliation</r:AttributeKey>
          <r:AttributeValue>Widengren</r:AttributeValue>
        </r:UserAttributePair>
        <a:IndividualIdentification>
          <a:IndividualName>
            <a:LastFamily>Jerker</a:LastFamily>
            <a:FullName>
              <r:String>Jerker</r:String>
            </a:FullName>
          </a:IndividualName>
        </a:IndividualIdentification>
      </a:Individual>
    </a:OrganizationScheme>
  </g:ResourcePackage>
  <s:StudyUnit>
    <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.StudyUnit:2.0</r:URN>
    <r:UserID typeOfUserID="datasetIdentifier">doi-10-5281-zenodo-8006451</r:UserID>
    <r:Citation>
      <r:Title>
        <r:String xml:lang="en">Dataset to "Fluorescence Bar-coding and Flowmetry based on Dark State Transitions in Fluorescence Emitters"</r:String>
      </r:Title>
      <r:Creator>
        <r:CreatorReference>
          <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.Individual-0:2.0</r:URN>
          <r:TypeOfObject>Individual</r:TypeOfObject>
        </r:CreatorReference>
      </r:Creator>
      <r:Publisher>
        <r:PublisherName>
          <r:String xml:lang="sv">Kungliga Tekniska högskolan</r:String>
          <r:String xml:lang="en">Royal Institute of Technology</r:String>
        </r:PublisherName>
      </r:Publisher>
      <r:Publisher>
        <r:PublisherName>
          <r:String xml:lang="sv">Kungliga Tekniska högskolan</r:String>
          <r:String xml:lang="en">Royal Institute of Technology</r:String>
        </r:PublisherName>
      </r:Publisher>
      <r:PublicationDate>
        <r:SimpleDate>2023-06-05</r:SimpleDate>
      </r:PublicationDate>
      <r:InternationalIdentifier>
        <r:IdentifierContent>10.5281/zenodo.8006451</r:IdentifierContent>
        <r:ManagingAgency controlledVocabularyAgencyName="DOI">DOI</r:ManagingAgency>
      </r:InternationalIdentifier>
    </r:Citation>
    <r:Abstract>
      <r:Content xml:lang="en">This folder contains all raw data underlying the results presented in a manuscript, submitted for publication to Journal of Physical Chemistry B, and entitled:

Fluorescence Bar-coding and Flowmetry based on Dark State Transitions in Fluorescence Emitters

Authored by:

B. Demirbay‡, E. Sandberg‡, J. Piguet, J. Widengren*

Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova University Center 106 91 Stockholm, Sweden

‡Contributed equally.

*Corresponding Author:

 Email:  jwideng@kth.se, Phone: +46-8-7907813

The data files are grouped into the different techniques used to generate them, and refer to the figures/tables in the manuscript where the extracted results are presented.

ABSTRACT

Reversible dark state transitions in fluorophores represent a limiting factor in fluorescence-based ultrasensitive spectroscopy, is a necessary basis for fluorescence-based super-resolution imaging, but may also offer additional, largely orthogonal fluorescence-based readout parameters. In this work, we analyzed the blinking kinetics of Cyanine5 (Cy5) as a bar-coding feature distinguishing Cy5 from rhodamine fluorophores having largely overlapping emission spectra. First, fluorescence correlation spectroscopy (FCS) solution measurements on mixtures of free fluorophores and fluorophore-labeled small unilamellar vesicles (SUVs) showed that Cy5 could be readily distinguished from the rhodamines by its reversible, largely excitation-driven trans-cis isomerization. This was next confirmed by transient state (TRAST) spectroscopy measurements, determining the fluorophore dark state kinetics in a more robust manner, from how the time-averaged fluorescence intensity varies upon modulation of the applied excitation light. TRAST was then combined with wide-field imaging of live cells, whereby Cy5 and rhodamine fluorophores could be distinguished on a whole cell level, as well as in spatially resolved, multiplexed images of the cells. Finally, we established a microfluidic TRAST concept, and showed how different mixtures of free Cy5 and rhodamine fluorophores and corresponding fluorophore-labeled SUVs could be distinguished on-the-fly when passing through a microfluidic channel. In contrast to FCS, TRAST does not rely on single-molecule detection conditions or a high time-resolution and is thus broadly applicable on different biological samples. Therefore, we expect that the bar-coding concept presented in this work can offer an additional, useful strategy for fluorescence-based multiplexing, implementable on a broad range of both stationary and moving samples.</r:Content>
    </r:Abstract>
    <r:Coverage>
      <r:TopicalCoverage>
        <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.TopicalCoverage:2.0</r:URN>
        <r:Subject xml:lang="en" controlledVocabularyID="103" controlledVocabularyName="Standard för svensk indelning av forskningsämnen 2025">Physical Sciences</r:Subject>
        <r:Subject xml:lang="sv" controlledVocabularyID="103" controlledVocabularyName="Standard för svensk indelning av forskningsämnen 2025">Fysik</r:Subject>
      </r:TopicalCoverage>
      <r:SpatialCoverage />
    </r:Coverage>
    <a:Archive>
      <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.Archive:2.0</r:URN>
      <a:ArchiveSpecific>
        <a:Item>
          <a:Access>
            <r:URN>urn:ddi:se.researchdata:doi-10-5281-zenodo-8006451.Archive-ArchiveSpecificType-AccessType:2.0</r:URN>
            <a:TypeOfAccess controlledVocabularyName="info:eu-repo-Access-Terms vocabulary">restrictedAccess</a:TypeOfAccess>
          </a:Access>
          <a:DataFileQuantity>0</a:DataFileQuantity>
        </a:Item>
      </a:ArchiveSpecific>
    </a:Archive>
  </s:StudyUnit>
</ddi:DDIInstance>