Fluorescence tools operating on a single-molecule scale

Discoveries in biosciences are frequently stimulated by the invention of new scientific tools. We like to push fluorescence techniques beyond the classic spatial and temporal resolution limits. Such low-invasive approaches offer the fascinating prospect of observing biomolecules in their native environment and understanding how they act in concert.

Our group focuses on the measurement and manipulation of inter- and intramolecular dynamics in a cellular setting. Tools for this purpose may be based on devices or (fluorescent) probes. A device-based approach we would like to highlight this year is a promising superlens-microscope design. An ongoing probe-based approach at our laboratory addresses the disputed question as to how protein binding may be affected by laser illumination itself and how these effects could be controlled in fluorescence imaging or even used as a tool.

A superlens assisted fluorescence microscope for surface imaging of biomolecules

Figure 1 (Click to view legend)

The concept of a “perfect” lens that is capable of resolving features smaller than the diffraction limit by amplifying the near-field was suggested nearly a decade ago (Pendry, 2000) and has intrigued the scientific community ever since. Obviously, such lenses would have far-reaching applications, particularly in fields such as nano-lithography, optical storage, and bio-imaging.

However, the production of materials or metamaterials with suitable properties and of suitable design has been hindered by practical problems such as material loss and surface roughness. Recent advances in the field have altered the situation: the production and application of a superlens-based microscope has become feasible in terms of technology. One design that may reduce some of these undesirable effects is the stacked metal-dielectric superlens. We are exploring the imaging ability of such a design for the specific purpose of imaging Green Fluorescent Protein (GFP) and other FP’s in the vicinity of the superlens surface. We recently found that a metallic/dielectric/metallic superlens may be suitable for imaging such fluorescent molecules with a deep sub-diffraction limit resolution (see Fig. 1). We are now working on fine tuning the parameters of the metallic and dielectric layers for imaging specific fluorophores. The proposed lens could be incorporated in a microscope setup and permit ultra-fast super-resolution surface imaging.

Towards the mechanism of photo-unbinding

Figure 2 (Click to view legend)

Fluorescent probes are commonly used in biological experiments. Despite their great success story over the last century, it is known that fluorescent conjugates can also influence the properties of the molecules under study. Our recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light; the same has been observed for protein-peptide binding, including toxins (see Fig. 2). This year, we succeeded in gaining insight into the mechanism of photounbinding by studying labeled calmodulin (CaM) and a set of CaM-binding peptides with different affinities to CaM. Our findings suggest that photounbinding is linked to photobleaching and a ‘radiative’ process requiring a fluorescent label. Interestingly, the photounbinding effect becomes stronger with increasing binding affinity, but does not induce breakage of covalent bonds. Our model is based on the assumption that an intermediate (transitional) complex is formed before the unbinding occurs. This is consistent with the labeled protein undergoing a conformational change which in turn is responsible for the unbinding.

 

We believe that knowledge of the involved molecular processes would not only lead to the systematic improvement of quantitative fluorescent studies, but also pave the way for inducing or inhibiting molecular interactions by light.
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