Author:

Keywords:

DNA, STED, stretched DNA, time gated STED

Abstract:

Characterization of nanomaterials by non-linair microscopy is increasing as it can shed new light on their properties and stimulated their usage. Here we use STED microscopy, a nanoscopy technique allowing circumventing the optical resolution limit. Deposition of matter is relevant to a plethora of applications and is of great importance in research. Hence, controlled deposition of nanomaterials and biomaterials, such as DNA, is of the interest of fundamental biophysical research and future applications, e.g. optical gene mapping. In this study we show the usefulness of time-gated STED to study DNA, instead of commonly used, slower investigation techniques. Our experimental parameters mimicked circumstances without any additional imaging buffers, as encountered in optical mapping. Evaporating droplets is an easy and fruitful way of depositing matter, in a controlled manner. Here we use droplets of fluorescent labelled DNA, on easy to prepare substrates that allow efficient binding of the biomolecules. Optimal DNA stretching is achieved by special translocation of the droplets; inducing a 'rolling flow'. This well studied system of YOYO-1 complexed with T7 dsDNA was deposited, as described above, and used in this new STED platform, which has orders of magnitude better temporal resolution than commonly used AFM techniques. By an in-house built STED system, these biophysical samples were investigated, confirming nanoscopy resolution (ca. 40 nm). Varying experimental parameters, STED enlightens the connecting spatial variations. Further investigation was done on the possible influence of the STED beam on the samples, via detailed microscopy and spectroscopy.