Two-photon direct laser writing (DLW) lithography is limited in the achievable structure size as well as in structure resolution. Adding stimulated emission depletion (STED) to DLW allowed overcoming both restrictions. We now push both to new limits. Using visible light for two-photon DLW (780 nm) and STED (532 nm), we obtain lateral structure sizes of 55 nm, a Sparrow limit of around 100 nm and we present two clearly separated lines spaced only 120 nm apart. The photo-resist used in these experiments is a mixture of tri- and tetra-acrylates and 7-Diethylamino-3-thenoylcoumarin as a photo-starter which can be readily quenched via STED.

Alternative splicing (AS) is an important regulatory process that leads to the creation of multiple RNA transcripts from a single gene. Alternative transcripts often carry premature termination codons (PTCs), which trigger nonsense-mediated decay (NMD), a cytoplasmic RNA degradation pathway. However, intron retention, the most prevalent AS event in plants, often leads to PTC-carrying splice variants that are insensitive to NMD; this led us to question the fate of these special RNA variants. Here, we present an innovative approach to monitor and characterize endogenous mRNA splice variants within living plant cells. This method combines standard confocal laser scanning microscopy for molecular beacon detection with a robust statistical pipeline for sample comparison. We demonstrate this technique on the localization of NMD-insensitive splice variants of two Arabidopsis thaliana genes, RS2Z33 and the SEF factor. The experiments reveal that these intron-containing splice variants remain within the nucleus, which allows them to escape the NMD machinery. Moreover, fluorescence recovery after photobleaching experiments in the nucleoplasm show a decreased mobility of intron-retained mRNAs compared with fully spliced RNAs. In addition, differences in mobility were observed for an mRNA dependent on its origin from an intron-free or an intron-containing gene.

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration—corresponding to only 39,000 molecules in the sample solution—and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 104 cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

We report here the development of a device for single-molecule imaging on large surface areas. A CCD camera operated in time delay and integration mode is synchronized with the movement of a sample scanning stage, enabling continuous data acquisition. Experiments on single fluorescent lipid molecules in supported lipid bilayers and on stained living cells demonstrate the capabilities of the method. Areas of up to 5 x 5 mm(2) were recorded within 11 min at a pixel size of 129 nm.

Telomeres comprise the protective caps of natural chromosome ends and function in the suppression of DNA damage signaling and cellular senescence. Therefore, techniques used to determine telomere length are important in a number of studies, ranging from those investigating telomeric structure to effects on human disease. Terminal restriction fragment (TRF) analysis has for a long time shown to be one of the most accurate methods for quantification of absolute telomere length and range from a number of species. As this technique centers on standard Southern blotting, telomeric DNA is observed on resulting autoradiograms as a heterogeneous smear. Methods to accurately determine telomere length from telomeric smears have proven problematic, and no reliable technique has been suggested to obtain mean telomere length values. Here, we present TeloTool, a new program allowing thorough statistical analysis of TRF data. Using this new method, a number of methodical biases are removed from previously stated techniques, including assumptions based on probe intensity corrections. This program provides a standardized mean for quick and reliable extraction of quantitative data from TRF autoradiograms; its wide application will allow accurate comparison between datasets generated in different laboratories.

Acrylate nanoanchors of subdiffraction-limited diameter are written with optical stimulated emission depletion (STED) lithography. After incubation, 98% of all nanoanchors are loaded quickly with fluorescently labeled antibodies. Controlling the size of the nanoanchors allows for limiting the number of the antibodies. Direct stochastic optical reconstruction microscopy (dSTORM) imaging, statistical distribution of fluorescence, quantitative fluorescence readout, and single molecule blinking consistently prove that 80% of the nanoanchors with a 65 nm diameter are carrying only one antibody each, which are functional as confirmed with live erythrocytes.

Abstract:We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and nonspecifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation.

Biomimetics is the interdisciplinary scientific field focused on the study and imitation of biological systems, with the aim of solving complex technological problems. In this paper, we present a new bio-inspired design for microneedles (MNs) and MN arrays, intended for rapidly coating the MNs with drug/vaccine. The biomimetic approach consists in ornamenting the lateral sides of pyramidal MNs with structures inspired by the external scent efferent systems of some European true bugs, which facilitate a directional liquid transport. To realize these MNs, two-photon polymerization (TPP) technique was used. Liquid coating capabilities of structured and non-structured MNs were compared. Moreover, both in-vivo and ex-vivo skin tests were performed to prove that MNs pierce the skin. We show that the arrays of MNs can be accurately replicated using a micro-moulding technique. We believe this design will be beneficial for the process of drug/vaccine loading onto the needles’ surfaces, by making it more efficient and by reducing the drug/vaccine wastage during MN coating process.Electronic supplementary materialThe online version of this article (10.1007/s10544-019-0456-z) contains supplementary material, which is available to authorized users.