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J-aggregates are fascinating fluorescent nanomaterials formed by highly ordered assembly of organic dyes with the spectroscopic properties dramatically different from that of single or disorderly assembled dye molecules. They demonstrate very narrow red-shifted absorption and emission bands, strongly increased absorbance together with the decrease of radiative lifetime, highly polarized emission and other valuable features. The mechanisms of their electronic transitions are understood by formation of delocalized excitons already on the level of several coupled monomers. Cyanine dyes are unique in forming J-aggregates over the broad spectral range, from blue to near-IR. With the aim to inspire further developments, this review is focused on the optical characteristics of J-aggregates in connection with the dye structures and on their diverse already realized and emerging applications.

The author discusses methods for the fluorometric determination of affinity constants by linear and nonlinear fitting methods. This is outlined in particular for the interaction between cyclodextrins and several anesthetic drugs including benzocaine. Special emphasis is given to the limitations of certain fits, and the impact of such studies on enzyme-substrate interactions are demonstrated. Both the experimental part and methods of analysis are well suited for students in an advanced lab.

The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc.4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.

The influence of an electron donating substituent on the photophysical properties of 1-azathioxanthone dyes has been investigated using optical spectroscopy and theoretical models. The motivation behind the study is based on the fact that thioxanthones are efficient triplet sensitizers, and thus promising sensitizers for lanthanide centered emission. By adding an aza group to one of the phenyl ring systems, direct coordination to a lanthanide center becomes possible, which makes azathoixanthones great candidates as antenna chromophores in lanthanide(III) based dyes. Here, three 1-azathioxanthone derivatives have been synthesized targeting efficient triplet formation following absorption in the visible range of the spectrum. This is achieved by adding methoxy groups to the 1-azathioxanthone core. The derivatives were characterized using absorption, emission, and time-gated emission spectroscopy, where fluorescent quantum yields, singlet and triplet excited states lifetimes were determined. The experimentally determined photophysical properties of the three 1-azathioxanthone compounds are contrasted to those of the parent thioxanthone and is rationalized using the Strickler–Berg equation, Hückel MO theory, and Dewar's rules in combination with computational chemistry. We find that the transition energies follow predictions, but that the overall photophysical properties are determined by the relative energies as well as the nature of the involved states in both the singlet and the triplet excited state manifolds.

Most accepted single particle tracking methods are able to obtain high-resolution trajectories for relatively short periods of time. In this work we apply a straightforward combination of single-particle tracking microscopy and metallic nanoparticles internalization on mouse chromaffin cells to unveil the intracellular trafficking mechanism of metallic-nanoparticle-loaded vesicles (MNP-V) complexes after clathrin dependent endocytosis. We found that directed transport is the major route of MNP-Vs intracellular trafficking after stimulation (92.6% of the trajectories measured). We then studied the MNP-V speed at each point along the trajectory, and found that the application of a second depolarization stimulus during the tracking provokes an increase in the percentage of low-speed trajectory points in parallel with a decrease in the number of high-speed trajectory points. This result suggests that stimulation may facilitate the compartmentalization of internalized MNPs in a more restricted location such as was already demonstrated in neuronal and neuroendocrine cells (Bronfman et al 2003 J. Neurosci.23 3209–20). Although further experiments will be required to address the mechanisms underlying this transport dynamics, our studies provide quantitative evidence of the heterogeneous behavior of vesicles mobility after endocytosis in chromaffin cells highlighting the potential of MNPs as alternative labels in optical microscopy to provide new insights into the vesicles dynamics in a wide variety of cellular environments.

Fluorescence anisotropy imaging microscopy (FAIM) measures the depolarization properties of fluorophores to deduce molecular changes in their environment. For successful FAIM, several design principles have to be considered and a thorough system-specific calibration protocol is paramount. One important calibration parameter is the G factor, which describes the system-induced errors for different polarization states of light. The determination and calibration of the G factor is discussed in detail in this article. We present a novel measurement strategy, which is particularly suitable for FAIM with high numerical aperture objectives operating in TIRF illumination mode. The method makes use of evanescent fields that excite the sample with a polarization direction perpendicular to the image plane. Furthermore, we have developed an ImageJ/Fiji plugin, AniCalc, for FAIM data processing. We demonstrate the capabilities of our TIRF-FAIM system by measuring $\beta$-actin polymerization in human embryonic kidney cells and in retinal neurons.

Here we describe a label-free detection strategy for large deletion mutation in breast cancer (BC) related gene BRCA1 based on a DNA-silver nanocluster (NC) fluorescence upon recognition-induced hybridization. The specific hybridization of DNA templated silver NCs fluorescent probe to target DNAs can act as effective templates for enhancement of AgNCs fluorescence, which can be used to distinguish the deletion of BRCA1 due to different fluorescence intensities. Under the optimal conditions, the fluorescence intensity of the DNA-AgNCs at emission peaks around 440 nm (upon excitation at 350 nm) increased with the increasing deletion type within a dynamic range from 1.0 × 10⁻¹⁰ to 2.4 × 10⁻⁶ M with a detection limit (LOD) of 6.4 × 10⁻¹¹ M. In this sensing system, the normal type shows no significant fluorescence; on the other hand, the deletion type emits higher fluorescence than normal type. Using this nanobiosensor, we successfully determined mutation using the non-amplified genomic DNAs that were isolated from the BC cell line.

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Ho³⁺/Yb³⁺/Tm³⁺/Li⁺:Gd₂(MoO₄)₃ nanophosphors successfully synthesised via solid state reaction method have been structurally and optically characterised. Under 980 nm diode laser excitation the nanophosphors emit intense blue, green, red and NIR emissions peaking at ∼476 nm, ∼543 nm, ∼646 nm and ∼798 nm corresponding to the ¹G₄ → ³H₆ (Tm³⁺), ⁵F₄, ⁵S₂ → ⁵I₈ (Ho³⁺), ⁵F_⁵ → ⁵I₈ (Ho³⁺) and ³H₄ → ³H₆ (Tm³⁺) transitions respectively. The upconversion emission intensity enhancement in the Ho³⁺–Yb³⁺–Tm³⁺–Li⁺:Gd₂(MoO₄)₃ nanophosphors for the green band is found to be ∼367, ∼50 and ∼9 times compared to the singly Ho³⁺ doped, Ho³⁺–Yb³⁺ co-doped and Ho³⁺–Yb³⁺–Tm³⁺ tri-doped Gd₂(MoO₄)₃ nanophosphors. The enhancement observed has been explained on the basis of energy transfer process and local field modifications around the rare earth ions. The energy transfer efficiency ∼5% is determined in the tridoped nanophosphors. The interaction involved between rare earth ions for energy transfer process is found to be dipole–dipole type. On changing the Tm³⁺ ions concentration the colour emitted from the tridoped nanophosphors is tuned from near white to blue region. In the tridoped nanophosphors, on varying the pump power the colour tunability has been observed.

2'-deoxyfuranouridine derivatives presenting various aryl groups have been synthesized through Cu(I)-catalyzed intramolecular cyclizations. Moreover, corresponding pyrrolo-dC derivatives have been synthesized and both families of compounds thoroughly characterized using UV/vis and fluorescence spectroscopy as well as time-dependent density functional theory calculations. The photophysical characterization, show that our newly synthesized derivatives of the important pyrrolo-dC family have high fluorescence quantum yields (QYs) and brightness values. Pyrrolo-dC derivative, 3a, shows an environment sensitive QY of up to >60% and brightness of almost 3000, in low polarity solvents and excitation and emission maxima between 365–381 nm and 479–510 nm, respectively, in solvents of different polarities. Two other derivatives, 3b and 3c, show high QYs and brightness values of up to 3300 that are fairly insensitive to their microenvironment. These promising photophysical features suggest future applicability as fluorescent nucleobase analogs.

Erbium (Er) doped zinc oxide (ZnO) powder was prepared using co-precipitation method. Effect of ytterbium (Yb) and molybdenum (Mo) co-doping on upconversion emission was studied. The emission intensity has been enhanced on Yb co-doping due to energy transfer from Yb to Er. Co-doping Mo in ZnO:Er, Yb has brought a significant enhancement in green upconversion emission, when excited with 980 nm laser. The enhancement in green emission on Mo doping is because of the formation of ${{\rm{Yb}}}^{3+}\mbox{--}{{{\rm{MoO}}}_{4}}^{2-}$ dimer. Since ZnO is a material having high thermal stability, the enhanced green emission in Mo, Er, Yb doped ZnO can be used for temperature sensor applications. The variation in relative intensity ratio of ²H_11/2 → ⁴I_15/2,⁴S_3/2 → ⁴I_15/2 transition can be used to sense the sample temperature.

In most cases, intermediate states of multistage folding proteins are not 'visible' under equilibrium conditions but are revealed in kinetic experiments. Time-resolved fluorescence spectroscopy was used in equilibrium denaturation studies. The technique allows for detecting changes in the conformation and environment of tryptophan residues in different structural elements of carbonic anhydrase II which in its turn has made it possible to study the intermediate states of carbonic anhydrase II under equilibrium conditions. The results of equilibrium and kinetic experiments using wild-type bovine carbonic anhydrase II and its mutant form with the substitution of leucine for alanine at position 139 (L139A) were compared. The obtained lifetime components of intrinsic tryptophan fluorescence allowed for revealing that, the same as in kinetic experiments, under equilibrium conditions the unfolding of carbonic anhydrase II ensues through formation of intermediate states.

Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid–solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (<0.1 mm²). Objective-based TIRFs are also expensive as they require dichroic mirrors and efficient notch filters to prevent specular reflection within the objective lenses. We have developed a compact 3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.

Fluorescence spectroscopy with an excitation-emission matrix (EEM) is a fast and inexpensive technique and has been applied to the detection of a very wide range of analytes. However, serious scattering and overlapping signals hinder the applications of EEM spectra. In this contribution, the multi-resolution capability of Tchebichef moments was investigated in depth and applied to the analysis of two EEM data sets (data set 1 consisted of valine-tyrosine-valine, tryptophan-glycine and phenylalanine, and data set 2 included vitamin B1, vitamin B2 and vitamin B6) for the first time. By means of the Tchebichef moments with different orders, the different information in the EEM spectra can be represented. It is owing to this multi-resolution capability that the overlapping problem was solved, and the information of chemicals and scatterings were separated. The obtained results demonstrated that the Tchebichef moment method is very effective, which provides a promising tool for the analysis of EEM spectra. It is expected that the applications of Tchebichef moment method could be developed and extended in complex systems such as biological fluids, food, environment and others to deal with the practical problems (overlapped peaks, unknown interferences, baseline drifts, and so on) with other spectra.