Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics
KAUST DepartmentLaboratory of DNA Replication and Recombination
Biological and Environmental Sciences and Engineering (BESE) Division
Permanent link to this recordhttp://hdl.handle.net/10754/552752
MetadataShow full item record
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.
CitationVersatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics 2011, 82 (11):113702 Review of Scientific Instruments
JournalReview of Scientific Instruments
- Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics.
- Authors: Ross J, Buschkamp P, Fetting D, Donnermeyer A, Roth CM, Tinnefeld P
- Issue date: 2007 Jan 18
- Single-molecule three-color FRET with both negligible spectral overlap and long observation time.
- Authors: Lee S, Lee J, Hohng S
- Issue date: 2010 Aug 19
- Red light, green light: probing single molecules using alternating-laser excitation.
- Authors: Santoso Y, Hwang LC, Le Reste L, Kapanidis AN
- Issue date: 2008 Aug
- Design and analysis of multi-color confocal microscopy with a wavelength scanning detector.
- Authors: Do D, Chun W, Gweon DG
- Issue date: 2012 May
- Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm.
- Authors: He L, Bradrick TD, Karpova TS, Wu X, Fox MH, Fischer R, McNally JG, Knutson JR, Grammer AC, Lipsky PE
- Issue date: 2003 May