Characterisation of optically driven microstructures for manipulating single DNA molecules under a fluorescence microscope

Abstract

Optical tweezers are powerful tools for manipulating single DNA molecules using fluorescence microscopy, particularly in nanotechnology-based DNA analysis. We previously proposed a manipulation technique using microstructures driven by optical tweezers that allows the handling of single giant DNA molecules of millimetre length that cannot be manipulated by conventional techniques. To further develop this technique, the authors characterised the microstructures quantitatively from the view point of fabrication and efficiency of DNA manipulation under a fluorescence microscope. The success rate and precision of the fabrications were evaluated. The results indicate that the microstructures are obtained in an aqueous solution with a precision ∼50 nm at concentrations in the order of 10(6) particles/ml. The visibility of these microstructures under a fluorescence microscope was also characterised, along with the elucidation of the fabrication parameters needed to fine tune visibility. Manipulating yeast chromosomal DNA molecules with the microstructures illustrated the relationship between the efficiency of manipulation and the geometrical shape of the microstructure. This report provides the guidelines for designing microstructures used in single DNA molecule analysis based on on-site DNA manipulation, and is expected to broaden the applications of this technique in the future.

Optically driven microtools with an antibody‐immobilised surface for on‐site cell assembly

“…These were trapped with optical tweezers, where the directions of their heights were aligned along the direction of the laser propagation, enabling us to define the orientation of the microtools simultaneously with a single beam trap. [ 13 , 14 , 15 , 23 ].…”

“…We previously reported the development of an enzyme‐immobilised microtool for the pinpoint processing of biomolecules, such as a chromosomal DNA molecule. [ 12 ] The microtool was fabricated with a negative photoresist SU‐8, which was driven by optical tweezers [ 13 , 14 , 15 ] and had an enzyme‐immobilised surface. SU‐8 allows for the fabrication of 3D microstructures with a high aspect ratio and is known to have high biocompatibility.…”

Driving Forces for Single DNA Stretching Assessed by In Situ TIRFM

“…Another vital aspect in DNA stretching studies is imaging methods for DNA molecules. In recent years, DNA molecules have been observed directly by high-resolution microscopic instruments, e.g., atomic force microscopy, ,, electron microscopy, and fluorescence microscopy. − Unlike the first two atomically precise methods that required complex equipment, accessible and facile optical methods, such as total internal reflection fluorescence microscopy (TIRFM), are also suitable for DNA imaging . More importantly, this nondestructive optical method offers an opportunity to study in situ DNA stretching, giving unique insights into behavior information of DNA molecules.…”

On-Site Processing of Single Chromosomal DNA Molecules Using Optically Driven Microtools on A Microfluidic Workbench

“…However, there are some limitations to these approaches, Microbeads have poor contact with target molecules because of their spherical shape [11]. Although an AFM tip allows for precise positioning, its integration with the imaging of a target sample remains technically challenging.…”

“…To address these issues, we previously developed optically driven microstructures for e cient DNA manipulation. These microstructures allow e cient contact between DNA molecules and a structure [11]. The technique is limited to the physical manipulation of molecules including picking up, winding, and unwinding of DNA molecules [12,13].…”