The TFAM-to-mtDNA ratio defines inner-cellular nucleoid populations with distinct activity levels

In human cells, generally a single mitochondrial DNA (mtDNA) is compacted into a nucleoprotein complex denoted the nucleoid. Each cell contains hundreds of nucleoids, which tend to cluster into small groups. It is unknown whether all nucleoids are equally involved in mtDNA replication and transcription or whether distinct nucleoid subpopulations exist. Here, we use multi-color STED super-resolution microscopy to determine the activity of individual nucleoids in primary human cells. We demonstrate that only a minority of all nucleoids are active. Active nucleoids are physically larger and tend to be involved in both replication and transcription. Inactivity correlates with a high ratio of the mitochondrial transcription factor A (TFAM) to the mtDNA of the individual nucleoid, suggesting that TFAM-induced nucleoid compaction regulates nucleoid replication and transcription activity in vivo. We propose that the stable population of highly compacted inactive nucleoids represents a storage pool of mtDNAs with a lower mutational load

Enhanced incorporation of subnanometer tags into cellular proteins for fluorescence nanoscopy via optimized genetic code expansion

With few-nanometer resolution recently achieved by a new generation of fluorescence nanoscopes (MINFLUX and MINSTED), the size of the tags used to label proteins will increasingly limit the ability to dissect nanoscopic biological structures. Bioorthogonal (click) chemical groups are powerful tools for the specific detection of biomolecules. Through the introduction of an engineered aminoacyl–tRNA synthetase/tRNA pair (tRNA: transfer ribonucleic acid), genetic code expansion allows for the site-specific introduction of amino acids with “clickable” side chains into proteins of interest. Welldefined label positions and the subnanometer scale of the protein modification provide unique advantages over other labeling approaches for imaging at molecular-scale resolution. We report that, by pairing a new N-terminally optimized pyrrolysyl–tRNA synthetase (chPylRS2020) with a previously engineered orthogonal tRNA, clickable amino acids are incorporated with improved efficiency into bacteria and into mammalian cells. The resulting enhanced genetic code expansion machinery was used to label β-actin in U2OS cell filopodia for MINFLUX imaging with minimal separation of fluorophores from the protein backbone. Selected data were found to be consistent with previously reported high-resolution information from cryoelectron tomography about the cross-sectional filament bundling architecture. Our study underscores the need for further improvements to the degree of labeling with minimal-offset methods in order to fully exploit molecularscale optical three-dimensional resolution

DNA-PAINT MINFLUX nanoscopy

MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, providing photon-efficient fluorophore localizations, has brought about three-dimensional resolution at nanometer scales. However, by using an intrinsic on–off switching process for single fluorophore separation, initial MINFLUX implementations have been limited to two color channels. Here we show that MINFLUX can be effectively combined with sequentially multiplexed DNA-based labeling (DNA-PAINT), expanding MINFLUX nanoscopy to multiple molecular targets. Our method is exemplified with three-color recordings of mitochondria in human cells

MINSTED nanoscopy enters the Ångström localization range

We report all-optical, room-temperature localization of fluorophores with precision in the Ångström range. These precisions are attained in a STED microscope, by encircling the fluorophore with the low-intensity edge of the STED donut beam, while constantly increasing the absolute donut power. Individual fluorophores bound to a DNA strand are localized with σ = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Since our experiments yield a localization precision σ = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up entirely new areas of application in the study of macromolecular complexes in cells

Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models

Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single-molecule localization microscopy with sub-10 nm spatial resolution in three dimensions. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation and the challenging nature of the data analysis. Here we report the development of a 4Pi-STORM microscope, which obtains optimal resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically while also using a simpler optical design. Dynamic spline PSF models incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for precision and minimizes phase-wrapping artifacts by making full use of the information content of the data. 4Pi-STORM achieves a near-isotropic three-dimensional localization precision of 2–3 nm, and we demonstrate its capa-bilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria

Gaia Data Release 1: Testing parallaxes with local Cepheids and RR Lyrae stars

Context. Parallaxes for 331 classical Cepheids, 31 Type II Cepheids, and 364 RR Lyrae stars in common between Gaia and the Hipparcos and Tycho-2 catalogues are published in Gaia Data Release 1 (DR1) as part of the Tycho-Gaia Astrometric Solution (TGAS). Aims. In order to test these first parallax measurements of the primary standard candles of the cosmological distance ladder, which involve astrometry collected by Gaia during the initial 14 months of science operation, we compared them with literature estimates and derived new period-luminosity (PL), period-Wesenheit (PW) relations for classical and Type II Cepheids and infrared PL, PL-metallicity (PLZ), and optical luminosity-metallicity (M V -[Fe/H]) relations for the RR Lyrae stars, with zero points based on TGAS. Methods. Classical Cepheids were carefully selected in order to discard known or suspected binary systems. The final sample comprises 102 fundamental mode pulsators with periods ranging from 1.68 to 51.66 days (of which 33 with σ Ω /Ω < 0.5). The Type II Cepheids include a total of 26 W Virginis and BL Herculis stars spanning the period range from 1.16 to 30.00 days (of which only 7 with σ Ω /Ω < 0.5). The RR Lyrae stars include 200 sources with pulsation period ranging from 0.27 to 0.80 days (of which 112 with σ Ω /Ω < 0.5). The new relations were computed using multi-band (V,I,J,K s ) photometry and spectroscopic metal abundances available in the literature, and by applying three alternative approaches: (i) linear least-squares fitting of the absolute magnitudes inferred from direct transformation of the TGAS parallaxes; (ii) adopting astrometry-based luminosities; and (iii) using a Bayesian fitting approach. The last two methods work in parallax space where parallaxes are used directly, thus maintaining symmetrical errors and allowing negative parallaxes to be used. The TGAS-based PL,PW,PLZ, and M V - [Fe/H] relations are discussed by comparing the distance to the Large Magellanic Cloud provided by different types of pulsating stars and alternative fitting methods. Results. Good agreement is found from direct comparison of the parallaxes of RR Lyrae stars for which both TGAS and HST measurements are available. Similarly, very good agreement is found between the TGAS values and the parallaxes inferred from the absolute magnitudes of Cepheids and RR Lyrae stars analysed with the Baade-Wesselink method. TGAS values also compare favourably with the parallaxes inferred by theoretical model fitting of the multi-band light curves for two of the three classical Cepheids and one RR Lyrae star, which were analysed with this technique in our samples. The K-band PL relations show the significant improvement of the TGAS parallaxes for Cepheids and RR Lyrae stars with respect to the Hipparcos measurements. This is particularly true for the RR Lyrae stars for which improvement in quality and statistics is impressive. Conclusions. TGAS parallaxes bring a significant added value to the previous Hipparcos estimates. The relations presented in this paper represent the first Gaia-calibrated relations and form a work-in-progress milestone report in the wait for Gaia-only parallaxes of which a first solution will become available with Gaia Data Release 2 (DR2) in 2018. © ESO, 2017

Rhodamine-Hoechst positional isomers for highly efficient staining of heterochromatin.

Hoechst conjugates to fluorescent dyes are popular DNA stains for live-cell imaging, but the relationship between their structure and performance remains elusive. This study of carboxyrhodamine–Hoechst 33258 conjugates reveals that a minimal change in the attachment point of the dye has dramatic effects on the properties of the final probe. All tested 6′-carboxyl dye-containing probes exhibited dual-mode binding to DNA and formed a dimmer complex at high DNA concentrations. The 5′-carboxyl dye-containing probes exhibited single-mode binding to DNA which translated into increased brightness and lower cytotoxicity. Up to 10-fold brighter nuclear staining by the newly developed probes allowed acquisition of stimulated emission depletion (STED) nanoscopy images of outstanding quality in living and fixed cells. Therefore we were able to estimate a diameter of ∼155 nm of the heterochromatin exclusion zones in the nuclear pore region in living cells and intact chicken erythrocytes and to localize telomeres relative to heterochromatin in living U-2 OS cells. Employing the highly efficient probes for two-color STED allowed visualization of DNA and tubulin structures in intact nucleated erythrocytes – a system where imaging is greatly hampered by high haemoglobin absorbance

Gpufit: An open-source toolkit for GPU-accelerated curve fitting.

We present a general purpose, open-source software library for estimation of non-linear parameters by the Levenberg-Marquardt algorithm. The software, Gpufit, runs on a Graphics Processing Unit (GPU) and executes computations in parallel, resulting in a significant gain in performance. We measured a speed increase of up to 42 times when comparing Gpufit with an identical CPU-based algorithm, with no loss of precision or accuracy. Gpufit is designed such that it is easily incorporated into existing applications or adapted for new ones. Multiple software interfaces, including to C, Python, and Matlab, ensure that Gpufit is accessible from most programming environments. The full source code is published as an open source software repository, making its function transparent to the user and facilitating future improvements and extensions. As a demonstration, we used Gpufit to accelerate an existing scientific image analysis package, yielding significantly improved processing times for super-resolution fluorescence microscopy datasets

Three dimensional live-cell STED microscopy at increased depth using a water immersion objective.

Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 mu m

Molecular contribution function in RESOLFT nanoscopy

The ultimate objective of a microscope of the highest resolution is to map the molecules of interest in the sample. Traditionally, linear imaging systems are characterized by their spatial frequency transfer function, which is given, in real space, by the point spread function (PSF). By extending the concept of the PSF towards the molecular contribution function (MCF), that quantifies the average contribution of a single fluorophore to the image, a straightforward concept for counting fluorophores is obtained. Using reversible saturable optical fluorescence transitions (RESOLFT), fluorophores are effectively activated only in a small, subdiffraction-sized volume before they are read out. During readout the signal exhibits an increased variance due to the stochastic nature of prior activation, which scales quadratically with the brightness of the active fluorophores while the mean of the signal scales only linearly with it. Using a two-state Markov model for the activation, showing comparable behavior to the switching kinetics of the switchable fluorescent protein rsEGFP2, we can approximate quantitatively the MCF of RESOLFT nanoscopy allowing to count the number of fluorophores within a subdiffraction-sized region of the sample. The method is validated on measurements of tubulin structures in Drosophila melagonaster larvae. Modeling and estimation of the MCF is a promising approach to quantitative microscopy