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.
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November 2011
Research Article|
November 07 2011
Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics
M. A. Sobhy;
M. A. Sobhy
1Laboratory of DNA Replication and Recombination, Division of Chemical and Life Sciences and Engineering,
King Abdullah University of Science and Technology
, Thuwal 23955, Saudi Arabia
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M. M. Elshenawy;
M. M. Elshenawy
a)
1Laboratory of DNA Replication and Recombination, Division of Chemical and Life Sciences and Engineering,
King Abdullah University of Science and Technology
, Thuwal 23955, Saudi Arabia
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M. Takahashi;
M. Takahashi
1Laboratory of DNA Replication and Recombination, Division of Chemical and Life Sciences and Engineering,
King Abdullah University of Science and Technology
, Thuwal 23955, Saudi Arabia
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B. H. Whitman;
B. H. Whitman
2
Laser Launch LLC
, York, Pennsylvania 17403, USA
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N. G. Walter;
N. G. Walter
3Department of Chemistry, Single-Molecule Analysis Group,
University of Michigan
, Ann Arbor, Michigan 48109, USA
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S. M. Hamdan
S. M. Hamdan
b)
1Laboratory of DNA Replication and Recombination, Division of Chemical and Life Sciences and Engineering,
King Abdullah University of Science and Technology
, Thuwal 23955, Saudi Arabia
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a)
Graduate Program in Biosciences.
b)
Author to whom correspondence should be addressed. Electronic mail: samir.hamdan@kaust.edu.sa.
Rev. Sci. Instrum. 82, 113702 (2011)
Article history
Received:
July 05 2011
Accepted:
October 06 2011
Citation
M. A. Sobhy, M. M. Elshenawy, M. Takahashi, B. H. Whitman, N. G. Walter, S. M. Hamdan; Versatile single-molecule multi-color excitation and detection fluorescence setup for studying biomolecular dynamics. Rev. Sci. Instrum. 1 November 2011; 82 (11): 113702. https://doi.org/10.1063/1.3657153
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