In situ investigation of biochemical and nanocolloidal dynamics using fast optical nanoscopy: Video rate high resolution fluorescence microscopy in material science and biochemistry.

Nanomaterials are all around us. They are widely used as they show some extraordinary properties as catalysts (showing unexpected reactivity or needing less starting material), as additives to change properties of bulk materials (exhibiting luminescent properties like in quantum dots, or altering wetting behavior for self cleaning effect), biomedical applications for target drug delivery, potential investigation tool or new treatment procedure. Most of these applications seem futuristic, yet nanomaterials are present in many household objects such as sunblock (sun burn protection), toothpaste (for improved cleansing and protection), in fridges (antimicrobial) and in computers (for miniaturization). To study their behavior, interaction and influence one needs to be able to visualize these materials, and their dynamics, at the real size.

For years biologists have used optical microscopy to study microscopic life, such as bacteria and viruses. Hence optical microscopy has proven itself as a convenient tool for studying microscale materials. Especially fluorescence microscopy, such as confocal microscopy, has been shown to be extremely useful in investigating dynamics of (sub)micron particles, with limiting disturbance of the sample. Nowadays, improvements in the field of optical microscopy, such as STED, allow even to obtain tens of nanometers in resolution. These techniques are classified as super-resolution fluorescence microscopy techniques or optical nanoscopy techniques.

As to control the location of the material of interest, droplets can be used. Droplets are a commonly encountered, three phase system (of liquid, gas and solid) and are or a side effect or a goal in many industrial and academic research.

Droplets are an easy way to assemble nano and biomaterials, in a controlled way.  This thesis aims to show the usefulness of fluorescence microscopy for studying sub-micron particles, of any origin, in a controllable model system: i.e. evaporating droplets.

The studies in this PhD deal with droplet induced substrate assembly of nanoparticles, microorganisms or DNA (a biomolecule well-known for carrying genetic information). Additional, supplementary information about the results is put at the end of this dissertation, in the respective appendices.

The well-known coffee ring effect was exploited. This is a flow inside a droplet that is present when it is pinned to a substrate and creating, upon drying, ring like deposits of the present suspended matter. Drops containing fluorescent bacteria and colloids, show this coffee ring flow. Upon addition of surfactants this flow profile changes, and subsequently also the final deposition. Remarkably, we see that bacteria that produce surfactants themselves show comparable flows and altered deposits. 

A follow-up study with a higher surfactant concentration showed a macroscopic repetitive assembly of the colloids is reached, along the droplet edge, and this in the shape of a flower.

In a third study, droplets containing fluorescent DNA were used to deposit linear fragments of DNA on a substrate. Tuning the substrate influences the efficiency of DNA retention and stretching. Further optimization, by droplet pulling, allowed efficient DNA linearization and binding, even at extremely low concentrations of DNA.

DNA can be linearly stretched and can reach lengths of micrometers. Yet DNA is 1D nanoscale system, as the base pairs only are a few nanometers wide. In the last study, tSTED (an extension of the high resolution STED technique) is used to investigate DNA assemblies on dry substrates. Varying buffer parameters allowed alternating DNA deposits, visual via tSTED in high resolution. Additional experiments showed that the DNA probe, YOYO-1 could interact with itself and deteriorate the image quality of STED. tSTED imaging allows us to overcome this deterioration and the life time information provides an additional resolution improvement.

This laserscanning tSTED platform will allow studying DNA stretching by droplets, in high spatial and temporal resolution, where other techniques would fail.