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Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or ‘iBALM’ can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling.

Direct visualization of DNA-protein interactions at the single-molecule level is of ever increasing importance to unravel the salient details of a wide range of DNA-associated processes. Recent emergence of super-resolution imaging has the potential to further boost the impact of such investigations. However, current imaging methods heavily rely on (fluorescent) labeling strategies, which can be challenging and potentially even interfere with the molecular interactions under scrutiny. Here, we introduce a new label-free method to image the presence and location of DNA-bound proteins with super-resolution on optically manipulated DNA. The method is based on localization microscopy of DNA-intercalating dyes that locally bind to bare DNA but not to protein-bound DNA sections, yielding an inverted image that reveals the ‘shadows’ of the proteins on the DNA: inverse binding-activated localization microscopy (iBALM). We present the proof of principle of iBALM as well as of functional variations to this method and provide experimental and theoretical data that describe the spatial and temporal resolution that can be obtained. iBALM has the potential to become a valuable addition to the single-molecule toolkit and enable direct visualization of processes that were previously not possible due to limitations posed by labeling.

Over the past two decades, single-molecule techniques have evolved into robust tools to study many fundamental biological processes. The combination of optical tweezers with fluorescence microscopy and microfluidics provides a powerful single-molecule manipulation and visualization technique that has found widespread application in biology. In this combined approach, the spatial (~ nm) and temporal (~ ms) resolution, as well as the force scale (~ pN) accessible to optical tweezers is complemented with the power of fluorescence microscopy. Thereby, it provides information on the local presence, identity, spatial dynamics, and conformational dynamics of single biomolecules. Together, these techniques allow comprehensive studies of, among others, molecular motors, protein–protein and protein–DNA interactions, biomolecular conformational changes, and mechanotransduction pathways. In this chapter, recent applications of fluorescence microscopy in combination with optical trapping are discussed. After an introductory section, we provide a description of instrumentation together with the current capabilities and limitations of the approaches. Next we summarize recent studies that applied this combination of techniques in biological systems and highlight some representative biological assays to mark the exquisite opportunities that optical tweezers combined with fluorescence microscopy provide.