Protein‐Specific,Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.     

Protein‐Specific,Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.     

Accreditation of proficiency-testing schemes in The Netherlands

Proficiency testing by laboratories, national accreditation bodies, and other third parties is becoming more and more considered as a standard and integral part of the quality control system. Therefore, it is of utmost importance that the quality of the provided proficiency-testing (PT) service is outstanding. If PT-schemes are set up in order to help laboratories monitor and improve their quality, PT-schemes need not only be of high quality themselves, but the organizer also needs to be able to demonstrate this. In The Netherlands formal accreditation of the organization of proficiency testing schemes is used as a tool to guarantee high quality schemes and also to enable organizers to demonstrate their competence. Since 1996, the Dutch Council for Accreditation (RvA) has used the ISO-Guide 43–1 to assess PT-organizers in The Netherlands. From January 2000, the ISO-guide 43–1 was replaced by the ILAC G13 document for assessing organizers. Up till now, four institutes have been accredited by the RvA for the organization of PT-schemes.     

Cholesterol degradation in archaeological pottery mediated by fired clay and fatty acid pro-oxidants

Cholesterol is generally absent in animal fat residues preserved in archaeological ceramic vessels. It is known from edible oil refining that during bleaching with activated clay sterols are degraded, largely via oxidation. Laboratory heating experiments using fired clay from replica pottery vessels promoted rapid degradation of cholesterol via oxidation. Furthermore, heating cholesterol with fatty acids (saturated and unsaturated) revealed additional degradation to occur independently of the ceramic matrix. As both conditions are met in archaeological pottery during animal (and plant) product processing involving heating, the very rare detection of sterols in organic residues can be explained.     

"Competitive quenching": a mechanism by which perihydroxylated perylenequinone photosensitizers can prevent adverse phototoxic damage caused by verteporfin during photodynamic therapy

Incorporation of photodynamic therapy into clinical practice for induction of vascular photo-occlusion highlights the need to prevent adverse phototoxicity to sensitive juxtaposed tissues, particularly in the retina. We developed a system termed "competitive quenching" to prevent adverse phototoxic damage. It involves differential compartmentalization of a photoactivator to the intravascular compartment for photoexcitation and delivery of phototoxicity to targeted vessels. A different photodynamic agent is partitioned to the extravascular retinal space to quench reactive oxygen species generated by photosensitization, thereby protecting the adjacent retinal tissues from adverse phototoxicity. The absorption spectra of quenchers must span wavelengths that are shorter and excluded from the spectral range of photoexcitation light to prevent photoactivation of the quencher. Perihydroxylated perylenequinones were found to be suitable to function as "competitive quenchers" with the prototype hypericin identified as a potent quencher. Here we examined the mechanisms operative in competitive quenching and suggest that hypericin forms a complex with verteporfin, thereby quenching singlet oxygen formation. Furthermore, we show that hypericin, with six phenolic hydroxyls, protects retinal and endothelial hybridoma cells from phototoxicity more effectively than the dimethyl tetrahydroxy helianthrone structural analog with only four such phenolic hydroxyls. The findings suggest that hydroxyl numbers contribute to the efficacy of competitive quenching.