Revolution in resolution: Abbe's resolution limit has been overcome in fluorescence microscopy by using light‐driven processes to switch the emission of fluorophores on and off. Alternatively, chemical reactions can be used, for example the coordination of Cu2+ ions to a fluorescent probe for the stochastic switching between spectroscopic states.

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

Transcription factors (TFs) are sequence-specific DNA-binding proteins [1] that control much of gene expression. TFs are natural biosensors and switches, translating chemical and physical signals (temperature shifts, light exposure, chemical concentrations, redox status) into transcriptional changes by modulating the binding of RNA polymerase to promoter DNA. Since changes in TF levels underlie fundamental biological processes such as DNA repair and cell-cycle progression, alterations in the levels of active TFs both lead to and indicate disease; for example, mutations in transcription factor p53 contribute to the rapid growth of cancer cells and, owing to their prevalence (p53 is mutated in roughly 50 % of all human tumors), they have served as cancer biomarkers.[2] Thus, methods for the sensitive detection and quantitation of TFs provide both fundamental information about gene regulation and a platform for diagnostics.TF detection often involves gel-based assays and Western blotting; although helpful in characterizing TF-DNA interactions, these assays are tedious, expensive, and qualitative, and consume large quantities of sample. Enzyme-linked immunosorbent assays (ELISAs) are more sensitive and offer higher throughput, but they require many preparation and signal-amplification steps for the detection of lowabundance TFs. Amplification is also required in the proximity-based ligation assay, [3] making it incompatible with TF detection in living cells and diagnostic settings that demand results within minutes.An additional TF detection assay is based on fluorescence resonance energy transfer (FRET) between two doublestranded DNA (dsDNA) fragments containing fluorescently labeled single-stranded complementary overhangs ("molecular beacons"). [4][5][6] In the presence of TF, the DNAs associate, resulting in donor fluorophore quenching as a result of FRET. This assay still requires significant amounts of sample and cannot detect low-abundance TFs; and because of the short dynamic range of FRET (1-10 nm), it also requires close proximity among the fluorophore, the quencher, and the protein-DNA interface, increasing the likelihood of steric interference with protein-DNA binding and complicating sensor design. Moreover, placing the fluorophore and the quencher on either side of the protein-binding site (usually 15-30 base pairs (bp) in length) on DNA results in very low FRET signals for most TFs.Here, we use alternating-laser excitation (ALEX) spectroscopy [7,8] to detect TFs and small molecules by means of the TF-dependent coincidence of fluorescently labeled DNA. Like the molecular-beacon assay, our method is based on TFdriven DNA association, is rapid, and requires no amplification. However, our assay can detect pm levels of TFs in small amounts of sample, and it is FRET-independent, bypassing the need to optimize fluorophore position or know the structural details of TF-DNA binding; this flexibility in labeling ensures unperturbed TF-DNA binding. Using ALEX, we demonstrate TF and small-molecule detection, assay m...

Near Zero Energy and Positive Energy communities are expected to play a significant part in EU’s strategy to cut greenhouse gas emissions by 2050. Within this context, the work presented in this paper aims to investigate the feasibility of: (a) a new-built positive energy neighborhood; and (b) the retrofit of an existing neighborhood to near zero energy performance in the city of Alexandroupolis, Greece. Proposed measures involve the rollout at the community scale of renewable energy technologies (PV, geothermal heat pump), energy efficiency (fabric insulation, district heating and cooling networks) and storage systems (batteries). A parametric analysis is conducted to identify the optimum combination of technologies through suitable technical and financial criteria. Results indicate that zero and near zero emissions targets are met with various combinations that impose insulation levels, according to building regulations or slightly higher, and consider renewable energy production with an autonomy of half or, more commonly, one day. In addition, the advantages of performing nearly zero energy retrofit at the district, rather than the building level, are highlighted, in an attempt to stimulate interest in community energy schemes.