Flat-top TIRF illumination boosts DNA-PAINT imaging and quantification

Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary 'blinking' without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super-resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy

124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.We thank Martin Spitaler and the imaging facility of the MPI of Biochemistry for confocal imaging support

Toward Absolute Molecular Numbers in DNA-PAINT

Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future

Investigations of Ra+^+ properties to test possibilities of new optical frequency standards

The present work tests the suitability of the narrow transitions $7s \ ^2S_{1/2} \to 6d ^2D_{3/2}$and$7s ^2S_{1/2} \to 6d ^2D_{5/2}$in Ra$^+$for optical frequency standard studies. Our calculations of the lifetimes of the metastable$6d$states using the relativistic coupled-cluster theory suggest that they are sufficiently long for Ra$^+$ to be considered as a potential candidate for an atomic clock. This is further corroborated by our studies of the hyperfine interactions, dipole and quadrupole polarizabilities and quadrupole moments of the appropriate states of this system.Comment: Latex files, 5 pages, 1 figur

Visualizing the Coupling between Red and Blue Stark States Using Photoionization Microscopy

In nonhydrogenic atoms in a dc electric field, the finite size of the ionic core introduces a coupling between quasibound Stark states that leads to avoided crossings between states that would otherwise cross. Near an avoided crossing, the interacting states may have decay amplitudes that cancel each other, decoupling one of the states from the ionization continuum. This well- known interference narrowing effect, observed as a strongly electric field- dependent decrease in the ionization rate, was previously observed in several atoms. Here we use photoionization microscopy to visualize interference narrowing in helium atoms, thereby explicitly revealing the mechanism by which Stark states decay. The interference narrowing allows measurements of the nodal patterns of red Stark states, which are otherwise not observable due to their intrinsic short lifetime

Lamb shift in muonic helium ion

The Lamb shift (2P_{1/2}-2S_{1/2}) in the muonic helium ion (mu ^4_2He)^+ is calculated with the account of contributions of orders alpha^3, alpha^4, alpha^5 and alpha^6. Special attention is given to corrections of the electron vacuum polarization, the nuclear structure and recoil effects. The obtained numerical value of the Lamb shift 1379.028 meV can be considered as a reliable estimate for the comparison with experimental data.Comment: 18 pages, 11 figure

Timing molecular motion and production with a synthetic transcriptional clock

The realization of artificial biochemical reaction networks with unique functionality is one of the main challenges for the development of synthetic biology. Due to the reduced number of components, biochemical circuits constructed in vitro promise to be more amenable to systematic design and quantitative assessment than circuits embedded within living organisms. To make good on that promise, effective methods for composing subsystems into larger systems are needed. Here we used an artificial biochemical oscillator based on in vitro transcription and RNA degradation reactions to drive a variety of “load” processes such as the operation of a DNA-based nanomechanical device (“DNA tweezers”) or the production of a functional RNA molecule (an aptamer for malachite green). We implemented several mechanisms for coupling the load processes to the oscillator circuit and compared them based on how much the load affected the frequency and amplitude of the core oscillator, and how much of the load was effectively driven. Based on heuristic insights and computational modeling, an “insulator circuit” was developed, which strongly reduced the detrimental influence of the load on the oscillator circuit. Understanding how to design effective insulation between biochemical subsystems will be critical for the synthesis of larger and more complex systems