Biology subjects

Bokhobza, A. F. E.

Publications and source records attributed to Bokhobza, A. F. E..

3 recordsLinked to original sources

bioRxiv2026

Characterizing MINFLUX imaging performance with DNA origami

MINFLUX, a second-generation super-resolution technique, can localize fluorescent markers approaching single-nanometer precision in three dimensions. Similar to previous super-resolution methodologies, the extended duration of acquisitions can result in drift that needs accurate correction to enable proper analysis and interpretation of the collected data. Here, we use DNA origami, housing sites of known spatial distribution fitted with repeat-domain docking strands for DNA-PAINT imaging to characterize imaging performance over extended duration MINFLUX acquisitions (6-20 h). Repeat-domain docking strands overcome site-loss and reveal residual drift in prolonged MINFLUX 3D acquisitions that we correct with an algorithm exploiting time-correlated shifts of localizations around identified DNA origami sites. Following correction of residual drift the site precision, i.e. the scatter of localizations around sites, is [~]2 nm in all directions. Comparison of site precision from extended repeat-domain docking strands with site precision from standard short 8-10 nucleotide docking strands exhibits no detectable loss of site precision. By adding DNA origami structures to mounted biological samples we apply our approach to the imaging of the cardiac ryanodine receptor 2 in cryosectioned heart tissue. The data suggests that for these protein targets single domain markers with repeat domain docking strands may be directly used for residual drift correction, simplifying sample preparation and acquisition protocols.

biophysics↗

bioRxiv2025

Optimizing effective labeling efficiency in MINFLUX 3D DNA-PAINT microscopy by maximizing marker detection probability.

MINFLUX is a powerful single-molecule approach capable of achieving high spatially isotropic resolution in three dimensions. Current implementations collect localizations strictly serially, but criteria for when to terminate acquisition are often unclear. We therefore systematically investigate the time course of effective labeling efficiency (ELE) and achievable saturation values in MINFLUX 3D DNA-PAINT microscopy of Nup96 proteins in a U-2 OS-Nup96-mEGFP modified cell line using a commercial MINFLUX microscope. ELE was measured with a maximum-likelihood template fitting assisted quantitative procedure. We collected data measured over various scan sizes and achieved ELE values of [~]60% after passing a time interval dependent on the region size, typically requiring long-duration acquisitions over several hours. Our data and a simple model suggest that maximizing marker detection is key to achieving the limits set by chemical labeling efficiency. A factor limiting the marker detection probability when using conventional DNA-PAINT markers is docking strand site-loss, observed over the duration required to build up the image data of MINFLUX acquisitions, which also limits the achievable number of labeling site visits to values around 1-3. Using repeat DNA-PAINT, i.e. employing oligonucleotide sequences with repeated docking sites, we observed greatly reduced site-loss and could increase the number of individual visits to site locations by more than threefold over the same period. Additionally, this enabled increasing stringency criteria for labeling (i.e. higher threshold values) and maximizing marker detection probabilities so that ELE reaches the limits set by chemical labeling efficiency.

biophysics↗

bioRxiv2024

Reduced non-specific binding of super-resolution DNA-PAINT markers by Shielded DNA-PAINT labeling protocols

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events [~]5 fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific background.

biophysics↗