Recent advances in the design and measurement capabilities of optical tweezers instruments, and especially the combination with multi-color fluorescence detection, have accommodated a dramatic increase in the versatility of optical trapping. Quadruple (Q)-trap optical tweezers are an excellent example of such an advance, by providing three-dimensional control over two constructs and thereby enabling for example DNA-DNA braiding. However, the implementation of fluorescence detection in such a Q-trapping system poses several challenges: (1) since typical samples span a distance in the order of tens of micrometers, it requires imaging of a large field of view, (2) in order to capture fast molecular dynamics, fast imaging with single-molecule sensitivity is desired, (3) in order to study three-dimensional objects, it could be needed to detect emission light at different axial heights while keeping the objective lens and thus the optically trapped microspheres in a fixed position. In this chapter, we describe design guidelines for a fluorescence imaging module on a Q-trap system that overcomes these challenges and provide a step-by-step description for construction and alignment of such a system. Finally, we present detailed instructions for proof-of-concept experiments that can be used to validate and highlight the capabilities of the instruments.
CITATION STYLE
Meijering, A. E. C., Bakx, J. A. M., Man, T., Heller, I., Wuite, G. J. L., & Peterman, E. J. G. (2022). Implementation of 3D Multi-Color Fluorescence Microscopy in a Quadruple Trap Optical Tweezers System. In Methods in Molecular Biology (Vol. 2478, pp. 75–100). Humana Press Inc. https://doi.org/10.1007/978-1-0716-2229-2_5
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