Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics

Handle URI:
http://hdl.handle.net/10754/552752
Title:
Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics
Authors:
Sobhy, M. A.; Elshenawy, M. M.; Takahashi, Masateru; Whitman, B. H.; Walter, N. G.; Hamdan, S. M.
Abstract:
Single-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation and detection is a key experimental feature that is under continuous development. In this paper, we describe in detail the design and the construction of a sophisticated and versatile multi-color excitation and emission fluorescence instrument for studying biomolecular dynamics at the single-molecule level. The setup is novel, economical and compact, where two inverted microscopes share a laser combiner module with six individual laser sources that extend from 400 to 640 nm. Nonetheless, each microscope can independently and in a flexible manner select the combinations, sequences, and intensities of the excitation wavelengths. This high flexibility is achieved by the replacement of conventional mechanical shutters with acousto-optic tunable filter (AOTF). The use of AOTF provides major advancement by controlling the intensities, duration, and selection of up to eight different wavelengths with microsecond alternation time in a transparent and easy manner for the end user. To our knowledge this is the first time AOTF is applied to wide-field total internal reflection fluorescence (TIRF) microscopy even though it has been commonly used in multi-wavelength confocal microscopy. The laser outputs from the combiner module are coupled to the microscopes by two sets of four single-mode optic fibers in order to allow for the optimization of the TIRF angle for each wavelength independently. The emission is split into two or four spectral channels to allow for the simultaneous detection of up to four different fluorophores of wide selection and using many possible excitation and photoactivation schemes. We demonstrate the performance of this new setup by conducting two-color alternating excitation single-molecule fluorescence resonance energy transfer (FRET) and a technically challenging four-color FRET experiments on doubly labeled duplex DNA and quadruple-labeled Holliday junction, respectively.
KAUST Department:
Laboratory of DNA Replication and Recombination; Biological and Environmental Sciences and Engineering (BESE) Division
Citation:
Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics 2011, 82 (11):113702 Review of Scientific Instruments
Publisher:
AIP Publishing
Journal:
Review of Scientific Instruments
Issue Date:
7-Nov-2011
DOI:
10.1063/1.3657153
Type:
Article
ISSN:
00346748
Additional Links:
http://scitation.aip.org/content/aip/journal/rsi/82/11/10.1063/1.3657153
Appears in Collections:
Articles; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorSobhy, M. A.en
dc.contributor.authorElshenawy, M. M.en
dc.contributor.authorTakahashi, Masateruen
dc.contributor.authorWhitman, B. H.en
dc.contributor.authorWalter, N. G.en
dc.contributor.authorHamdan, S. M.en
dc.date.accessioned2015-05-14T07:01:32Zen
dc.date.available2015-05-14T07:01:32Zen
dc.date.issued2011-11-07en
dc.identifier.citationVersatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics 2011, 82 (11):113702 Review of Scientific Instrumentsen
dc.identifier.issn00346748en
dc.identifier.doi10.1063/1.3657153en
dc.identifier.urihttp://hdl.handle.net/10754/552752en
dc.description.abstractSingle-molecule fluorescence imaging is at the forefront of tools applied to study biomolecular dynamics both in vitro and in vivo. The ability of the single-molecule fluorescence microscope to conduct simultaneous multi-color excitation and detection is a key experimental feature that is under continuous development. In this paper, we describe in detail the design and the construction of a sophisticated and versatile multi-color excitation and emission fluorescence instrument for studying biomolecular dynamics at the single-molecule level. The setup is novel, economical and compact, where two inverted microscopes share a laser combiner module with six individual laser sources that extend from 400 to 640 nm. Nonetheless, each microscope can independently and in a flexible manner select the combinations, sequences, and intensities of the excitation wavelengths. This high flexibility is achieved by the replacement of conventional mechanical shutters with acousto-optic tunable filter (AOTF). The use of AOTF provides major advancement by controlling the intensities, duration, and selection of up to eight different wavelengths with microsecond alternation time in a transparent and easy manner for the end user. To our knowledge this is the first time AOTF is applied to wide-field total internal reflection fluorescence (TIRF) microscopy even though it has been commonly used in multi-wavelength confocal microscopy. The laser outputs from the combiner module are coupled to the microscopes by two sets of four single-mode optic fibers in order to allow for the optimization of the TIRF angle for each wavelength independently. The emission is split into two or four spectral channels to allow for the simultaneous detection of up to four different fluorophores of wide selection and using many possible excitation and photoactivation schemes. We demonstrate the performance of this new setup by conducting two-color alternating excitation single-molecule fluorescence resonance energy transfer (FRET) and a technically challenging four-color FRET experiments on doubly labeled duplex DNA and quadruple-labeled Holliday junction, respectively.en
dc.publisherAIP Publishingen
dc.relation.urlhttp://scitation.aip.org/content/aip/journal/rsi/82/11/10.1063/1.3657153en
dc.rightsArchived with thanks to Review of Scientific Instrumentsen
dc.titleVersatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamicsen
dc.typeArticleen
dc.contributor.departmentLaboratory of DNA Replication and Recombinationen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalReview of Scientific Instrumentsen
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionDepartment of Chemistry, Single-Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan 48109, USAen
dc.contributor.institutionLaser Launch LLC, York, Pennsylvania 17403, USAen
kaust.authorSobhy, Mohamed Abdelmabouden
kaust.authorHamdan, Samiren
kaust.authorElshenawy, Mohameden
kaust.authorTakahashi, Masateruen
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