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.
Abstract
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 imaging of DNA origami structures.
Single Molecule Localization Microscopy (SMLM) currently attains a lateral resolution of around 10 nm approaching molecular size. Together with increasingly specific fluorescent labeling, it opens ...the possibility to quantitatively analyze molecular organization. When the labeling density is high enough, SMLM provides clear images of the molecular organization. However, either due to limited labeling efficiency or due to intrinsically low molecular abundance, SMLM delivers a small set of sparse and highly precise localizations. In this work, we introduce a correlation analysis of molecular locations based on the functional dependence of the complementary cumulative distribution function (CCDF) of the distance to the first neighbor (
r
1
). We demonstrate that the log(−log(CCDF(
r
1
)))
vs.
log(
r
1
) is characterized by a scaling exponent
n
that takes extreme values of 2 for a random 2D distribution and 1 for a strictly linear arrangement, and find that
n
is a robust and sensitive metric to distinguish characteristics of the underlying structure responsible for the molecular distribution, even at a very low labeling density. The method enables the detection of fibrillary organization and the estimation of the diameter of host fibers under conditions where a visual inspection provides no clue.
We introduce a robust and sensitive metric to characterize the underlying structure responsible for the molecular distribution, even at a very low labeling density.
Localization of single fluorescent emitters is central for many 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 constituted a breakthrough for achieving a ∼10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Since then, other related methods have been developed and a common framework has been established for single-molecule localization through sequential structured illumination. Now, fluorescence nanoscopy and tracking of single fluorophores with true nanometric resolution are sitting on solid conceptual and experimental ground. There is an ample scope of questions where they can be applied in the near future to gain new insight into the dynamics and photophysics of natural and synthetic systems at the nanometer scale.
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.
Based on functional evidence, we have previously demonstrated that early ventral Notch1 activity restricts dorsoanterior development in
We found that Notch1 has ventralizing properties and abolishes ...the dorsalizing activity of β-catenin by reducing its steady state levels, in a process that does not require β-catenin phosphorylation by glycogen synthase kinase 3β. In the present work, we demonstrate that Notch1 mRNA and protein are enriched in the ventral region from the beginning of embryogenesis in
This is the earliest sign of ventral development, preceding the localized expression of
,
and Ventx genes in the ventral center and the dorsal accumulation of nuclear β-catenin. Knockdown experiments indicate that Notch1 is necessary for the normal expression of genes essential for ventral-posterior development
These results indicate that during early embryogenesis ventrally located Notch1 promotes the development of the ventral center. Together with our previous evidence, these results suggest that ventral enrichment of Notch1 underlies the process by which Notch1 participates in restricting nuclear accumulation of β-catenin to the dorsal side.
The developmental brain gene NPAS3 stands out as a hot spot in human evolution because it contains the largest number of human-specific, fast-evolving, conserved, non-coding elements. In this paper ...we studied 2xHAR142, one of these elements that is located in the fifth intron of NPAS3. Using transgenic mice, we show that the mouse and chimp 2xHAR142 orthologues behave as transcriptional enhancers driving expression of the reporter gene lacZ to a similar NPAS3 expression subdomain in the mouse central nervous system. Interestingly, the human 2xHAR142 orthologue drives lacZ expression to an extended expression pattern in the nervous system. Thus, molecular evolution of 2xHAR142 provides the first documented example of human-specific heterotopy in the forebrain promoted by a transcriptional enhancer and suggests that it may have contributed to assemble the unique properties of the human brain.
The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula ...organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear β-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that
expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both,
and
(a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of
cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in
in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.
The establishment of the embryonic dorsoventral axis in
occurs when the radial symmetry around the egg's animal-vegetal axis is broken to give rise to the typical symmetry of Bilaterians. We have ...previously shown that the Notch1 protein is ventrally enriched during early embryogenesis in
and zebrafish and exerts ventralizing activity through β-Catenin destabilization and the positive regulation of ventral center genes in
. These findings led us to further investigate when these asymmetries arise. In this work, we show that the asymmetrical distribution of Notch1 protein and mRNA precedes cortical rotation and even fertilization in
Moreover, we found that in unfertilized eggs transcripts encoded by the ventralizing gene
are also asymmetrically distributed in the animal hemisphere and
transcripts accumulate consistently on the same side of the eccentric maturation point. Strikingly, a Notch1 asymmetry orthogonal to the animal-vegetal axis appears during
oogenesis. Thus, we show for the first time a maternal bias in the distribution of molecules that are later involved in ventral patterning during embryonic axialization, strongly supporting the hypothesis of a dorsoventral prepattern or intrinsic bilaterality of
eggs before fertilization.
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