Sobhy M A, Elshenawy M M, Takahashi M, Whitman B H, Walter N G, Hamdan S M
Laboratory of DNA Replication and Recombination, Division of Chemical and Life Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia.
Rev Sci Instrum. 2011 Nov;82(11):113702. doi: 10.1063/1.3657153.
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.
单分子荧光成像处于用于研究体外和体内生物分子动力学的工具前沿。单分子荧光显微镜进行同步多色激发和检测的能力是一项关键实验特性，目前仍在不断发展。在本文中，我们详细描述了一种用于在单分子水平研究生物分子动力学的精密且通用的多色激发和发射荧光仪器的设计与构建。该装置新颖、经济且紧凑，两台倒置显微镜共享一个带有六个独立激光源的激光组合模块，这些激光源的波长范围从400纳米到640纳米。尽管如此，每台显微镜都能独立且灵活地选择激发波长的组合、顺序和强度。这种高灵活性是通过用声光可调滤光器（AOTF）取代传统机械快门来实现的。AOTF的使用带来了重大进展，它能以透明且便于终端用户操作的方式，在微秒级交替时间内控制多达八种不同波长的强度、持续时间和选择。据我们所知，这是AOTF首次应用于宽场全内反射荧光（TIRF）显微镜，尽管它已在多波长共聚焦显微镜中普遍使用。来自组合模块的激光输出通过两组每组四根单模光纤耦合到显微镜，以便能够独立优化每个波长的TIRF角度。发射光被分成两个或四个光谱通道，以允许同时检测多达四种不同的荧光团，这些荧光团种类繁多，并可采用多种可能的激发和光激活方案。我们分别通过对双标记双链DNA进行双色交替激发单分子荧光共振能量转移（FRET）实验，以及对四重标记霍利迪连接体进行技术上具有挑战性的四色FRET实验，展示了这种新装置的性能。
