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

The Subcolonization and Buildup of \u3ci\u3eTetrastichus Julis,\u3c/i\u3e (Hymenoptera: Eulophidae) a Larval Parasitoid of the Cereal Leaf Beetle, (Coleoptera: Chrysomelidae) in the Lower Peninsula of Michigan

Following initial establishment of the parasitoid, Tetrastichus julis (Walker), at a carefully managed field nursery, releases of parasitized Oulema melanopus larvae were made by Michigan county agents at preselected sites throughout the lower peninsula during 1970-74. A follow-up recovery program during 1971-75 revealed continued dispersion and population increase for T. julis. An independent census verified the increasing rates of parasitism

Effects of Neighboring Nectar-Producing Plants on Populations of Pest Lepidoptera and Their Parasitoids in Broccoli Plantings

Eggs and larvae of the imported cabbageworm, Pieris rapae, were much more abundant in broccoli interplanted with nectar-producing plants than in broccoli monoculture. More diamondback moth larvae, Plutella xylostella, occurred in broccoli interplanted with or adjacent to nectar-producing plants than in broccoli monoculture. Density of cabbage looper larvae, Trichoplusia ni, was similar among the three types of broccoli plantings. For Cotesia rubecula, established in Michigan after introduction from Yugoslavia, pupae were more numerous in broccoli interplanted with nectar-producing plants than in other plots. High parasitism rates of diamondback moth, mainly by Diadegma insulare, were observed in every plot, but there were no differences in parasitism of diamondback moth between the treatments. Results indicate that the interactions between pests, parasitoids and nectar-producing plants are complex and may be different for each species

Intended and Unintended Effects of Youth Bicycle Helmet Laws

Over 20 states have adopted laws requiring youths to wear a helmet when riding a bicycle. We confirm previous research indicating that these laws reduced fatalities and increased helmet use, but we also show that the laws significantly reduced youth bicycling. We find this result in standard two-way fixed effects models of parental reports of youth bicycling, as well as in triple difference models of self-reported bicycling among high school youths that explicitly account for bicycling by youths just above the helmet law age threshold. Our results highlight important intended and unintended consequences of a well-intentioned public policy.

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

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.Published as part of The Journal of Physical Chemistry virtual special issue “W. E. Moerner Festschrift”