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. 2020 Apr 6;48(6):e34.
doi: 10.1093/nar/gkaa061.

Imaging unlabeled proteins on DNA with super-resolution

Anna E C Meijering  1 , Andreas S Biebricher  1 , Gerrit Sitters  1 , Ineke Brouwer  1 , Erwin J G Peterman  1 , Gijs J L Wuite  1 , Iddo Heller  1
Affiliations

Imaging unlabeled proteins on DNA with super-resolution

Anna E C Meijering et al. Nucleic Acids Res. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Inverse microscopy proof of principle. (A) Schematic of inverse microscopy principle. Inverse microscopy relies on the competition of binding sites between protein (grey oval) and fluorescent probe (rod). (B) YOPRO imaging in the absence of hRAD51 reveals a homogeneously stained DNA strand (exposure time: 1 s). (C) Fluorescence wide-field images of hRAD51-Alexa647 signal (upper, acquired in absence of YOPRO) and the subsequently acquired YOPRO image (lower) on DNA that is tethered between two optically trapped polystyrene beads (exposure time: 1s). (D) Intensity profile between the white dotted lines of the wide-field images of (C) shows a strong correlation between the position of peaks in the hRAD51-A647 image (green profile) and the dips in the YOPRO image (black profile).
Figure 2.
Figure 2.
Super-resolution imaging of DNA with BALM and visualization of DNA-bound proteins with iBALM. (A) Fluorescence images show individual SxO binding events on optically manipulated DNA (exposure time: 1 s). (B) Super-resolution reconstruction of the SxO binding events on optically manipulated DNA. (C) Cross section and Gaussian fit taken perpendicular to the DNA orientation, as indicated in the super-resolved image in (B). (D) Wide-field image of hRAD51-A647 filaments bound to DNA (upper) and the corresponding reconstructed super-resolution image of SxO binding to DNA (lower) accumulated over 100 minutes. (E) Intensity profile along the DNA between the white dotted lines of the images in (C) show a strong correlation between hRAD51 position (green profile) and dark spots in the super-resolved SxO image (black profile). (F) Comparison of the intensity profile of the diffraction-limited RAD51 signal and the inverted intensity profile of the iBALM signal, at the position indicated by the arrows in (D) and (E). The dashed lines are gaussian fits that correspond to FWHM values of formula image and formula image nm for the hRAD51-A647 and SxO signals, respectively.
Figure 3.
Figure 3.
Monte Carlo simulations give insight into the performance of iBALM. (A) Example of a simulated intensity profile with a localization density formula image. Red area depicts the blocked region representing a protein patch. Green region is used to determine mean and standard deviation of the intensity profile. (B) Influence of the threshold value on the probability of finding false positives (black) and on the minimal detectable patch size (red). Three different combinations of localization densities were compared, while keeping the average number of photons constant at 100 per nm (continuous, dashed and dotted line). (C) The probability of detecting a protein patch of varying size as a function of localization density. Color scale depicts probability of detecting true positives. The threshold parameter and number of photons per intercalator were chosen to be formula image and formula image respectively. (D) Probability of detecting small protein patches (<10 nm) at a photon yield of formula image as a function of localization density.
Figure 4.
Figure 4.
Inverse microscopy applications. (A) YOPRO imaging reveals a sparse binding pattern after the DNA was saturated with hRAD51 binding (exposure time: 1 s). (B) Intensity profile of the fluorescence image of (A). Black triangles indicate the position of detected peaks. Inset: histogram of nearest peak distances. (C) Kymograph of eGFP-labeled hXLF on optically manipulated DNA (line trace: 190 ms). DNA-bound hXLF-eGFP oligomers were bleached and are visualized as dark traces (arrows) in a brighter background of transiently binding hXLF-eGFP monomers.
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