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On behalf of the Editorial Board of Methods and Applications in Fluorescence and IOP Publishing we are delighted to invite you to read the first articles in our new journal. Methods and Applications in Fluorescence is forged out of the renowned MAF conference series of the same name and we fully expect the natural synergy between the two to provide the ideal platform for moving the field of fluorescence forward.

Our aim is for this new journal to reflect the truly global and diverse impact fluorescence is having across many disciplines and help fluorescence achieve its full potential. Just as MAF is the leading conference in fluorescence we are confident of the high impact of this new journal.

Methods and Applications in Fluorescence has a distinguished Editorial Board that is drawn from the MAF conference Permanent Steering Committee. Together with the Editorial Board and the rest of the community, the journal will closely track the very latest developments in fluorescence while delivering a fair and constructive review process.

We are very pleased that this journal is backed by the Institute of Physics, one of the world's premier learned societies. IOP Publishing has a wealth of experience in science publishing that dates back to 1874. It is a not-for-profit organization that publishes over 60 journals, many on multidisciplinary topics and many including seminal contributions from Nobel Laureates. Any funding surplus generated by IOP Publishing goes directly back into science through the Institute of Physics, thus helping to nurture science for future generations.

We invite submissions as regular articles, review articles and technical notes within the scope of the journal, which includes all the major aspects of fluorescence. This covers both theory and experiment across spectroscopy, imaging, materials, labels, probes and sensors. The applications of fluorescence to emerging areas in bionanotechnology, nanotechnology and medicine are very much part of the vision for the journal.

Methods and Applications in Fluorescence is a journal for the whole community, so do please join us in helping to give the field of fluorescence the standing it deserves by submitting your latest and most exciting work. The success of the journal depends on all of us in the field and we look forward to working with you on this exciting project.

David Birch, Yves Mély and Otto S Wolfbeis Editors in Chief

Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.

Ensemble fluorescence decays are usually analyzed with a sum of exponentials. However, broad continuous distributions of lifetimes, either unimodal or multimodal, occur in many situations. A simple and flexible fitting function for these cases that encompasses the exponential is the Becquerel function. In this work, the applicability of the Becquerel function for the analysis of complex decays of several kinds is tested. For this purpose, decays of mixtures of four different fluorescence standards (binary, ternary and quaternary mixtures) are measured and analyzed. For binary and ternary mixtures, the expected sum of narrow distributions is well recovered from the Becquerel functions analysis, if the correct number of components is used. For ternary mixtures, however, satisfactory fits are also obtained with a number of Becquerel functions smaller than the true number of fluorophores in the mixture, at the expense of broadening the lifetime distributions of the fictitious components. The quaternary mixture studied is well fitted with both a sum of three exponentials and a sum of two Becquerel functions, showing the inevitable loss of information when the number of components is large. Decays of a fluorophore in a heterogeneous environment, known to be represented by unimodal and broad continuous distributions (as previously obtained by the maximum entropy method), are also measured and analyzed. It is concluded that these distributions can be recovered by the Becquerel function method with an accuracy similar to that of the much more complex maximum entropy method. It is also shown that the polar (or phasor) plot is not always helpful for ascertaining the degree (and kind) of complexity of a fluorescence decay.

We report the use of photonic crystal fibres (PCF) as spectrofluorimetric systems in which sample solutions are excited within the microstructure of the fibre. The use of intra-fibre excitation has several advantages that combine to enable highly sensitive measurements of fluorescence spectra and lifetimes: long path-lengths are achieved by the efficient guidance of the fundamental mode; sample volumes contained within the micron-scale structure are very small, only a few nanolitres per cm of path; collection and guidance of the emitted fluorescence is efficient and the fluorescence lifetime is unperturbed. Fluorophores in bulk solution can be studied in hollow core PCF, whereas the use of PCF with a suspended, solid core enables selective excitation of molecules in close proximity to the silica surface, through interaction with the evanescent field. We demonstrate the measurement of fluorescence spectra and fluorescence lifetimes in each of these excitation regimes and report the detection of attomole quantities of fluorescein.

Structured illumination microscopy (SIM) and time-resolved confocal fluorescence microscopy are applied to investigate the nanomorphology of thin films comprising typical blends of the conjugated polymer, poly (3-hexylthiophene) (P3HT), and [6, 6]-phenyl C₆₁-butyric acid methyl ester (PCBM), used for organic photovoltaic applications. SIM provides evidence for the presence of a thin emissive region around the crystalline regions of PCBM and at the tips of rod-like domains. The time-resolved measurements show that the emission surrounding the PCBM rods is longer lived than the bulk of the film. The two modes of microscopy provide complementary evidence indicating that electron–hole separation is inhibited between the polymer and the large PCBM-rich domains in these regions. We show here that structured illumination microscopy is a viable method of gaining additional information from these photovoltaic materials, despite their weak emission.

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method for characterizing the mode of motion of nanocarriers and for quantifying their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP–DOPC/DNA (DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOPC: dioleoylphosphocholine) and DC-Chol–DOPE/DNA (DC-Chol: 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol; DOPE: dioleoylphosphatidylethanolamine) lipoplexes in CHO-K1 (CHO: Chinese hamster ovary) live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient: D ≈ 0.003 μm² s⁻¹). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity v ≈ 0.03 μm² s⁻¹ and random motion. The method permitted us to generate an intracellular transport map showing several regions of concerted motion of lipoplexes.

The development of Alzheimer's disease is associated with the aggregation of the beta-amyloid peptides Aβ₁₋₄₀ and Aβ₁₋₄₂. It is believed that the small oligomers formed during the early stages of the aggregation are neurotoxic and involved in the process of neurodegeneration. In this paper we use fluorescence decay measurements of beta-amyloid intrinsic fluorophore tyrosine (Tyr) and molecular dynamics (MD) simulations to study the early stages of oligomer formation for the Aβ₁₋₄₀ and Aβ₁₋₄₂ peptides in vitro. We demonstrate that the lifetime distributions of the amyloid fluorescence decay efficiently describe changes in the complex Tyr photophysics during the peptide aggregation and highlight the differences in aggregation performance of the two amyloids. Tyr fluorescence decay is found to be a more sensitive sensor of Aβ₁₋₄₀ aggregation than Aβ₁₋₄₂ aggregation. The MD simulation of the peptide aggregation is compared with the experimental data and supports a four-rotamer model of Tyr.

One-bead one-compound combinatorial library beads exhibit varying levels of autofluorescence after solid phase combinatorial synthesis. Very often this causes significant problems for automated on-bead screening using TentaGel beads and fluorescently labeled target proteins. Herein, we present a method to overcome this limitation when fluorescence activated bead sorting is used as the screening method. We have equipped the COPAS bead sorting instrument with a high-speed profiling unit and developed a spectral autofluorescence correction method. The correction method is based on a simple algebraic operation using the fluorescence data from two detection channels and is applied on-the-fly in order to reliably identify hit beads by COPAS bead sorting. Our method provides a practical tool for the fast and efficient isolation of hit beads from one-bead one-compound library screens using either fluorescently labeled target proteins or biotinylated target proteins. This method makes hit bead identification easier and more reliable. It reduces false positives and eliminates the need for time-consuming pre-sorting of library beads in order to remove autofluorescent beads.