Biology subjects

Acuna, G. P.

Publications and source records attributed to Acuna, G. P..

5 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↗

bioRxiv2024

Multiplexed lifetime imaging of single molecules with a gated single-photon camera

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512x512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Forster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.

biophysics↗

bioRxiv2024

Super-Resolution Simplified: Sub-10nm Imaging Over Large Areas and Deep Penetration via SDC-OPR and DNA-PAINT

Single Molecule Localization Microscopy (SMLM) has traditionally faced challenges to optimize signal-to-noise ratio, penetration depth, field-of-view (FOV), and spatial resolution simultaneously. Here, we show that DNA-PAINT imaging on a Spinning Disk Confocal with Optical Photon Reassignment (SDC-OPR) system overcomes these trade-offs, enabling high-resolution imaging across multiple cellular layers and large FOVs. We demonstrate the systems capability with DNA origami constructs and biological samples, including nuclear pore complexes, mitochondria, and microtubules, achieving a spatial resolution of 6 nm in the basal plane and sub-10 nm localization precision at depths of 9 {micro}m within a 53 x 53 {micro}m{superscript 2} FOV. Additionally, imaging of the developing Drosophila eye epithelium at depths up to 9 {micro}m with sub-13 nm average localization precision, reveals distinct E-cadherin populations in adherens junctions. Quantitative analysis of Collagen IV deposition in this epithelium indicated an average of 46 {+/-} 27 molecules per secretory vesicle. These results underscore the versatility of DNA-PAINT on an SDC-OPR for advancing super-resolution imaging in complex biological systems.

biophysics↗

bioRxiv2024

Direct single-molecule detection and super-resolution imaging with a low-cost portable smartphone-based microscope

We present a novel, low-cost, portable smartphone-based fluorescence microscope capable of directly detecting single molecules without signal amplification. The setup leverages the image sensors and data handling capacity of mass-produced smartphones, making it adaptable to any smartphone and capable of detecting single molecules across the visible spectral range. We showcase this capability through single-molecule measurements on DNA origami models and super-resolution microscopy of biological cells by single-molecule localization microscopy. This development paves the way for biotechnology innovations making use of massively distributed or personalized assays with single-molecule sensitivity with the potential to revolutionize digital bioassays, point-of-care diagnostics, field expeditions, STEM outreach, and life science education.

biophysics↗

bioRxiv2020

Addressable Nanoantennas with Cleared Hotspots for Single-Molecule Detection on a Portable Smartphone Microscope

The advent of highly sensitive photodetectors1,2 and the development of photostabilization strategies3 made detecting the fluorescence of a single molecule a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g. in point-of-care diagnostic settings, where costly equipment would be prohibitive.4 Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in the NACHOS emit up to 461-fold brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia "on the road ".

biophysics↗