Papers by Fernando D. Stefani
Contributions to Zoology, 2007
The phylogenetic relationships of the scleractinian genus Psammocora with the other genera tradit... more The phylogenetic relationships of the scleractinian genus Psammocora with the other genera traditionally included in the family Siderastreidae and some Fungiidae are assessed based on combined skeletal and molecular data. P. explanulata differs from the other examined congeneric species (P. contigua, P. digitata, P. nierstraszi , P. profundacella, P. superficialis, and P. stellata) in possessing interstomatous septa between adult corallites, costae, and in having continuous buttress-like structures joining septal faces (i.e., fulturae) which typically occur in fungiids. These characters are shared with Coscinaraea wellsi but not with the remainder of the examined siderastreids (the congeneric C. columna, and Anomastraea irregularis, Horastrea indica, Pseudosiderastrea tayamai, Siderastrea savignyana) whose septa are interconnected by typical synapticulae. Most of the examined species form septa with distinct transverse groups of centers of calcification, a biomineralization pattern ...
Cornell University - arXiv, Nov 28, 2022
FRET-based approaches are a unique tool for sensing the immediate surroundings and interactions o... more FRET-based approaches are a unique tool for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and FLIM (Fluorescence Lifetime Imaging Microscopy) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, we demonstrate an approach to obtain super-resolved FRET imaging based on singlemolecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope. DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) with fluorogenic probes provides a suitable combination of background reduction and blinking kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.
Light: Science & Applications
After publication of this article 1 , it was brought to our attention that two of the Supplementa... more After publication of this article 1 , it was brought to our attention that two of the Supplementary Information "List of potentially interested researchers" and "news article" were attached by mistake, they should be removed. The original publication has been corrected. Reference 1. Masullo et al. An alternative to MINFLUX that enables nanometer resolution in a confocal microscope. Light Sci. Appl. 11, 199 (2022).
Light: Science & Applications
Using sequential excitation with a minimum of light to localize single fluorescent molecules repr... more Using sequential excitation with a minimum of light to localize single fluorescent molecules represented a breakthrough because it delivers 1–2 nm precision with moderate photon counts, enabling tracking and super-resolution imaging with true molecular resolution. Expanding this concept to multi-photon regimes may be a useful complement to reach even higher localization precision and get deeper into biological specimens.
Light: Science & Applications
Localization of single fluorescent emitters is key for physicochemical and biophysical measuremen... more Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1–2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imagi...
Nano Letters, Jan 13, 2021
We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule local... more We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.
Nano Letters, Feb 15, 2011
Optical antennas link objects to light. Here, we analyze metal nanorod antennas as cavities with ... more Optical antennas link objects to light. Here, we analyze metal nanorod antennas as cavities with variable reflection coefficients to derive the interaction of dipolar transitions with radiation through the antenna modes. The presented analytical model accurately describes the complete emission process, and is summarized in a phase-matching equation. We show how antenna modes evolve as they become increasingly more bound, i.e. plasmonic. The results illustrate why efficient antennas should not be too plasmonic, and how subradiant even modes can evolve into weakly-interacting dark modes. Our description is valid for the interaction of nanorods with light in general, and is thus widely applicable.
Computer Physics Communications, 2022
Focused optical fields are key to a multitude of applications involving light-matter interactions... more Focused optical fields are key to a multitude of applications involving light-matter interactions, such as optical microscopy, single-molecule spectroscopy, optical tweezers, lithography, or quantum coherent control. A detailed vectorial characterization of the focused optical fields that includes a description beyond the paraxial approximation is key to optimize technological performance as well as for the design of meaningful experiments and interpret properly their results. Here, we present PyFocus, an open-source Python software package to perform fully vectorial calculations of focused electromagnetic fields after modulation by an arbitrary phase mask and in the presence of a multilayer system. We provide a graphical user interface and high-level functions to easily integrate PyFocus into custom scripts. Furthermore, to demonstrate the potential of PyFocus, we apply it to extensively characterize the generation of toroidal foci with a high numerical aperture objective, as it is commonly done in super-resolution fluorescence microscopy methods such as STED or MINFLUX. We provide examples of the effects of different experimental factors such as polarization, aberrations, and misalignments of key optical elements. Finally, we present calculations of toroidal foci through an interface of different mediums, and, to our knowledge, the first calculations of toroidal foci generated in total internal reflection conditions.
run_SIMPLER is a graphical user interface that runs in Matlab (version 2015a or later with Curve ... more run_SIMPLER is a graphical user interface that runs in Matlab (version 2015a or later with Curve Fitting Tool installed). This app allows users of SIMPLER to perform all necessary operations to decode the axial positions of single molecules directly from 2D-SMLM-TIRF data. This is the first release of the software.
The Journal of Chemical Physics, 2022
While colloidal chemistry provides ways to obtain a great variety of nanoparticles with different... more While colloidal chemistry provides ways to obtain a great variety of nanoparticles with different shapes, sizes, material composition, and surface functions, their controlled deposition and combination on arbitrary positions of substrates remains a considerable challenge. Over the last ten years, optical printing arose as a versatile method to achieve this purpose for different kinds of nanoparticles. In this article, we review the state of the art of optical printing of single nanoparticles and discuss its strengths, limitations, and future perspectives, by focusing on four main challenges: printing accuracy, resolution, selectivity, and nanoparticles photostability. KEYWORDS: single nanoparticle, nanocrystal array, thermo osmosis, optical forces Colloidal chemistry allows the preparation of nanoparticles (NPs) with different compositions, sizes, and morphologies, which in turn define their unique physical and chemical properties that are impossible to obtain in bulk materials. In order to study them at the single-particle level and to take advantage of their features in circuits and devices, it is necessary to develop methods to bring the colloidal NPs from the liquid phase to specific locations of solid substrates. However, obtaining arbitrary arrays of colloidal NPs with different compositions, morphologies, and functions is still an open challenge. 1
Neuromethods, 2018
The aggregation of proteins into amyloid fibrils is related to more than 30 diseases, including t... more The aggregation of proteins into amyloid fibrils is related to more than 30 diseases, including the most common neurodegenerative conditions. Amyloid fibrillation is a nucleation-dependent polymerization reaction where monomeric protein first assembles into oligomers that in turn serve as nuclei for fibril formation. Recently, nanoparticles of various compositions and sizes have been investigated as nucleation centers for amyloid fibrillation. The interaction of nanoparticles with amyloid proteins can generate intermediate structures able to accelerate or inhibit fibrillation, and therefore, they constitute a tool to control and manipulate amyloid fibrillation which may be the key to elucidate molecular mechanisms or to devise therapies. In this chapter, we first give a general overview about the use of nanoparticles as artificial nucleation centers for amyloid aggregation, and then we focus on gold nanoparticles providing detailed protocols for their functionalization and use in amyloid fibrillation assays. 1. Amyloid fibrillation as a nucleation and growth polymerization. 2. Nanoparticles as nucleation centers. 3. Unique properties of gold nanoparticles. 4. Fabrication and surface modification of gold nanoparticles. 5. Amyloid aggregation assays with gold nanoparticles. 6. Protocols.
Biophysical Reports, 2022
Localization of single fluorescent molecules is key for physicochemical and biophysical measureme... more Localization of single fluorescent molecules is key for physicochemical and biophysical measurements such as single-molecule tracking and super-resolution imaging by singlemolecule localization microscopy (SMLM). Recently a series of methods have been developed in which the localization precision is enhanced by interrogating the molecular position with a sequence of spatially modulated patterns of light. Among them, the MINFLUX technique outstands for achieving a ~10-fold improvement compared to wide-field camera-based singlemolecule localization, reaching ~1 − 2 nm localization precision at moderate photon counts. Here, we present a common mathematical framework for this type of measurement that allows a fair comparison between reported methods and facilitates the design and evaluation of new methods. With it, we benchmark all reported methods for single-molecule localization using sequential structured illumination, including long-established methods such as orbital tracking, along with two new proposed methods: orbital tracking and raster scanning with a minimum of intensity.
The detection of individual molecules has found widespread application in molecular biology, phot... more The detection of individual molecules has found widespread application in molecular biology, photochemistry, polymer chemistry, quantum optics and super-resolution microscopy. Tracking of an individual molecule in time has allowed identifying discrete molecular photodynamic steps, action of molecular motors, protein folding, diffusion, etc. down to the picosecond level. However, methods to study the ultrafast electronic and vibrational molecular dynamics at the level of individual molecules have emerged only recently. In this review we present several examples of femtosecond single molecule spectroscopy. Starting with basic pump–probe spectroscopy in a confocal detection scheme, we move towards deterministic coherent control approaches using pulse shapers and ultra-broad band laser systems. We present the detection of both electronic and vibrational femtosecond dynamics of individual fluorophores at room temperature, showing electronic (de)coherence, vibrational wavepacket interfere...
Table of Contents SI Experimental Section 1-Computational chemistry 2-Synthesis and characterizat... more Table of Contents SI Experimental Section 1-Computational chemistry 2-Synthesis and characterization 3-Cell lines culture and plasmids, conditions for measurements in cells 4-Super-resolution fluorescence microscopy. STORM imaging SI Results 1-Computational chemistry 2-On the synthetic strategy of ABP-09 3-Solvatochromism of ABP-09 4-STORM performance of ABP-09 in cells 5-Experiments in cells and estimation of the CRHR1-ABP-09 binding constant References SI Experimental Section 1: Computational chemistry. During ligand preparation, partial atomic charges were taken from the MMFF force field [1-5]. Hydrogen atoms were added to the receptor structure using ICM, followed by a local energy minimization in the torsional space. Asp and Glu side chains were assigned a −1 charge, and all Arg and Lys side chains were assigned a +1 charge. Histidine tautomers were selected according to their hydrogen bonding pattern. In docking, the receptor was represented by six energy maps, namely, those corresponding to
Biophysical Reviews, 2021
Fluorescence nanoscopy represented a breakthrough for the life sciences as it delivers 20-30 nm r... more Fluorescence nanoscopy represented a breakthrough for the life sciences as it delivers 20-30 nm resolution using far-field fluorescence microscopes. This resolution limit is not fundamental but imposed by the limited photostability of fluorophores under ambient conditions. This has motivated the development of a second generation of fluorescence nanoscopy methods that aim to deliver sub-10 nm resolution, reaching the typical size of structural proteins and thus providing true molecular resolution. In this review, we present common fundamental aspects of these nanoscopies, discuss the key experimental factors that are necessary to fully exploit their capabilities, and discuss their current and future challenges.
Fil: Pellegrotti, Jesica Vanesa. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Ofic... more Fil: Pellegrotti, Jesica Vanesa. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisica; Argentina
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Papers by Fernando D. Stefani