Biology subjects

Zunino, A.

Publications and source records attributed to Zunino, A..

3 recordsLinked to original sources

bioRxiv2024

Array Detection Enables Large Localization Range for Simple and Robust MINFLUX

The MINFLUX concept significantly enhances the spatial resolution of single-molecule localization microscopy (SMLM) by overcoming the limit imposed by the fluorophores photon counts. Typical MINFLUX microscopes localize the target molecule by scanning a zero-intensity focus around the molecule in a circular trajectory, with smaller trajectory diameters yielding lower localization uncertainties for a given number of photons. Since this approach requires the molecule to be within the scanned trajectory, MINFLUX typically relies on a photon-demanding iterative scheme with decreasing trajectory diameters. Although the iterative procedure does not substantially reduce the photon efficiency of MINFLUX, this approach is prone to misplacements of the trajectory and increases the systems complexity. In this work, we introduce ISM-FLUX, a novel implementation of MINFLUX using image-scanning microscopy (ISM) with a single-photon avalanche diode (SPAD) array detector. ISM-FLUX provides precise MINFLUX localization within the trajectory while maintaining conventional photon-limited uncertainty outside it. The robustness of ISM-FLUX localization results in a larger localization range and greatly simplifies the architecture, which may facilitate broader adoption of MIN-FLUX.

bioengineering↗

bioRxiv2023

A Compact and Effective Photon-Resolved Image Scanning Microscope

Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative super-resolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data-acquisition system. Here, we present a data-acquisition module based on the digital frequency domain principle, able to record photons essential spatial and temporal features. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multi-species approach encoding the fluorophores excitation spectra in the time domain. Lastly, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, super-resolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques which effectively harness all the sample information encoded in each photon.

biophysics↗

bioRxiv2022

Focus-ISM for Sharp and Gentle Super-Resolved Microscopy

Super-resolution microscopy is routinely used for fixed and thin samples, while its feasibility for imaging live and thick samples is still limited. In the case of stimulated emission depletion (STED) microscopy, the high-intensity illumination required to achieve effective sub-diffraction resolution can introduce photo-damage, thus reducing the compatibility of the technique with live-cell imaging. Moreover, the out-of-focus fluorescence background may overcome the often faint signal stemming from the focal point, thus constraining imaging to thin samples. Here, we combined STED microscopy with image-scanning microscopy (ISM) to mitigate these limitations without any practical disadvantages. We first enhanced a laser scanning microscope (LSM) by introducing a detector array, hence providing access to a set of additional spatial information that is not available with a typical single-element detector. Then, we exploited this extended dataset to implement focus-ISM, a novel method that relaxes the high-intensity requirement of STED microscopy and removes the out-of-focus background. Additionally, we generalized the focus-ISM method to conventional LSM, namely without a STED beam. The proposed approach requires minimal architectural changes compared with conventional STED microscopes but provides substantial advantages for live and thick sample imaging while maintaining all compatibility with all recent advances in STED and confocal microscopy. As such, focus-ISM represents an essential step towards a universal super-resolved LSM technique for subcellular imaging.

biophysics↗