Widengren, J. | bioRxiv Science

bioRxiv2024

Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking

MINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with more relaxed fluorophore brightness and photostability requirements than for other SRM techniques. Nonetheless, low localization probabilities have been reported in several MINFLUX studies, and a broader use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. In this work, we identified fluorophore blinking as a main cause of erroneous (and dismissed) fluorophore localizations in MINFLUX imaging and devised strategies to overcome these effects. We systematically studied the blinking/switching properties of cyanine fluorophores emitting in the far-red or NIR range, and over typical time scales ({micro}s-10ms), sample and excitation conditions used in MINFLUX imaging. By subsequent simulations of representative MINFLUX localization procedures, we found that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. However, these localization errors could be suppressed by balanced redox buffers and repetitive excitation beam scans. Implementing these strategies, and replacing the slower, intrinsic switching of the fluorophores needed for the localization by transient binding of fluorophore-labelled DNA strands to complementary DNA strands attached to the targets (DNA-PAINT), we could for the first time demonstrate NIR-MINFLUX imaging with nanometer localization precision. This work presents an overall strategy, where fluorophore blinking characterization and subsequent simulations make it possible to design optimal sample and excitation conditions, opening for NIR-MINFLUX imaging, as well as for a broader use of fluorophores in MINFLUX and related SRM studies.

biophysics↗

bioRxiv2021

Coincident Fluorescence Burst Analysis of dUTP-Loaded Exosome-Mimetic Nanovesicles

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/463914v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13a85b2org.highwire.dtl.DTLVardef@11f6cd3org.highwire.dtl.DTLVardef@219a73org.highwire.dtl.DTLVardef@232f95_HPS_FORMAT_FIGEXP M_FIG C_FIG The targeting functionality and low immunogenicity of exosomes and exosome-mimetic nanovesicles make them promising as drug-delivery carriers. To tap into this potential, accurate non-destructive methods to load them and characterize their contents are of utmost importance. However, their small size, polydispersity and aggregation in solution make quantitative characterizations of their loading particularly challenging. Here we develop an ad-hoc methodology based on a burst analysis of dual-color confocal fluorescence microscopy experiments, suited for quantitative characterizations of exosome-like nanovesicles and of their loading. We apply it to study bioengineered nanovesicles, loaded with dUTP cargo molecules, synthetized from detergent-resistant membranes of animal extracellular vesicles and human red blood cells. For both classes of bioengineered nanovesicles we prove, by means of dual-color fluorescence cross-correlation spectroscopy (FCCS), successful loading. Furthermore, by a dual-color coincident fluorescence burst (DC-CFB) analysis of the experimental data, we retrieve size and loading statistics for both types of nanovesicles. The procedure affords single-vesicle characterizations, which are essential for reliable quantitative studies of loading processes in exosomes and exosome-mimetic nanovesicles, especially in light of the typically high heterogeneity of their populations. Moreover, the method implementation can be easily adapted to the investigation of a variety of combinations of different cargo molecules and biological nanovesicles besides the proof-of-principle demonstrations considered in this study. The results provide a powerful characterization tool, well-suited for the optimization of loading processes of biomimetic nanovesicles and their advanced engineering for therapeutic drug delivery.

biophysics↗