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Fluorescence spectroscopy and modeling provide powerful means to characterize biomacromolecular structures, dynamics, and interactions. Förster resonance energy transfer serves as a key technique for this due to its nanometer-scale distance sensitivity. Quantitative interpretation of fluorescence data relies on models that link molecular structure to observable spectroscopic quantities and vice versa. Integrative modelling frameworks combine fluorescence observables with complementary structural information to infer molecular structures and conformational ensembles. This review outlines conceptual components of fluorescence-based modeling, discusses dye representations, and highlights advances toward refined models enabling quantitative structural analysis. Finally, we discuss the prediction of spectroscopic properties of dyes based on biomolecular structures and fluorescence assay design beyond traditional FRET applications.

Near-infrared fluorescence imaging offers improved spatial precision by reducing light scattering and absorption in tissue. Despite this key advantage, the NIR region is limited by the availability of fluorophores, most of which exhibit relatively low quantum yield. In this study, gold nanospheres with absorption peaks in the visible range were used to enhance the fluorescence intensity of the cyanine NIR fluorophore IRdye 800 in the first NIR window of the electromagnetic spectrum. AuNSs with diameters ranging from 5 to 25 nm were chosen to investigate the impact of a nanoparticle size on fluorescence enhancement, functionalized with polyethylene glycol of varying molecular weights to optimize the distance between the fluorophore and the nanoparticle surface. Theoretical analyses using finite-difference time-domain simulations and experimental comparisons with non-metallic nanoparticles were performed to identify the factors contributing to the enhancement of fluorescence. PEGylated AuNSs conjugated with IRdye 800 (AuNDs) exhibited decreased photoisomerization, resulting in increased fluorescence intensity and altered fluorescence lifetimes. The observed enhancement in the fluores

Fluorescence-guided surgery has emerged as a vital tool for tumour resection procedures. As well as intraoperative tumour visualisation, 5-ALA-induced PpIX provides an avenue for quantitative tumour identification based on ratiometric fluorescence measurement. To this end, fluorescence imaging and fibre-based probes have enabled more precise demarcation between the cancerous and healthy tissues. These sensing approaches, which rely on collecting the fluorescence light from the tumour resection site and its remote spectral sensing, introduce challenges associated with optical losses. In this work, we demonstrate the viability of tumour detection at the resection site using a miniature fluorescence measurement system. Unlike the current bulky systems, which necessitate remote measurement, we have adopted a millimetre-sized spectral sensor chip for quantitative fluorescence measurements. A reliable measurement at the resection site requires a stable optical window between the tissue and the optoelectronic system. This is achieved using an antifouling diamond window, which provides stable optical transparency. The system achieved a sensitivity of 92.3% and specificity of 98.3% in detec

Variation in fluorescence intensity from rhodamine 6G dye was investigated. A small volume of dye solution was optically excited with a 0.4 mW, 532nm wavelength cw-laser light. The dye was dissolved in methanol and glycerol for a concentration of 10mg/ml. With the optical excitation, initially the fluorescence intensity was observed to rise, and then it decayed, along with a steady shift of fluorescence peak from 562 nm to 543 nm. The observation of initial enhancement in fluorescence from start to 7 minutes of excitation, can partly be due to the low excitation power, therefore slower rate of change of fluorescence intensity with time. Simulation studies indicate that the photo bleaching was taking place from all the energy states of the dye molecules, which is an extension of the concept that the photo bleaching takes place at the excited triplet state whereas the fluorescence takes place due to transition between ground and excited singlet states. A steady shift in fluorescence peak position, from 562 nm to 543 nm, was observed during the fluorescence life of the dye, at a rate of 0.0113 nm/s during fluorescence enhancement and 0.026 nm/s during photo bleaching.

We examine quantitatively the transition process from emitting to not-emitting states of fluorescent molecules with a machine learning technique. In a fluorescently labeled DNA, the fluorescence occurs continuously under irradiation, but it often transfers to the not-emitting state corresponding to a charge-separated state. The trajectory of the fluorescence consists of repetitions of light-emitting (ON) and not-emitting (OFF) states, called blinking, and it contains a very large amount of noise due to the several reasons, so in principle, it is difficult to distinguish the ON and OFF states quantitatively. The fluorescence trajectory is a typical stochastic process, and therefore requires advanced time-series data analysis. In the present study, we analyze the fluorescence trajectories using a hidden Markov model, and calculate the probability density of the ON and OFF duration. From the analysis, we found that the ON-duration probability density can be well described by an exponential function, and the OFF-duration probability density can be well described by a log-normal function, which are verified in terms of Kolmogorov-Smirnov test. The time-bin dependence in the fluorescence

This paper concerns an inverse problem for fluorescence diffuse optical tomography (FDOT) reconstructing locations of multiple point targets from the measured temporal response functions. The targets are multiple fluorescent point objects with a nonzero fluorescence lifetime at unknown locations. Peak time, when the temporal response function of the fluorescence reaches its maximum, is a robust parameter of the temporal response function in FDOT because it is most less suffered by the artifacts, such as noise, and is easily determined by experiments. We derive an approximate peak time equation based on asymptotic analysis in an explicit way in the case of nonzero fluorescence lifetime when there are single and multiple point targets. The performance of the approximation is numerically verified. Then, we develop a bisection algorithm to reconstruct the location of a single point target from the algorithm proposed in [4] for the case of zero fluorescence lifetime. Moreover, we propose a boundary-scan algorithm for the reconstruction of locations of multiple point targets. Finally, several numerical experiments are implemented to show the efficiency and robustness of the addressed alg

Analyses of spectral data often assume a linear mixing hypothesis, which states that the spectrum of a mixed substance is approximately the mixture of the individual spectra of its constituent parts. We evaluate this hypothesis in the context of dissolved organic matter (DOM) fluorescence spectroscopy for endmember abundance recovery from mixtures of three different DOM endmembers. We quantify two key sources of experimental variation, and statistically evaluate the linear mixing hypotheses in the context of this variation. We find that there is not strong statistical evidence against this hypothesis for high-fluorescence readings, and that true abundances of high-fluorescence endmembers are accurately recovered from the excitation-emission fluorescence spectra of mixed samples using linear methods. However, abundances of a low-fluorescence endmember are less well-estimated, in that the abundance coefficient estimates exhibit a high degree of variability across replicate experiments.

For the calorimetric determination of the primary energy of extensive air showers, measured by fluorescence telescopes, a precise knowledge of the conversion factor (fluorescence yield) between the deposited energy in the atmosphere and the number of emitted fluorescence photons is essential. The fluorescence yield depends on the pressure and the temperature of the air as well as on the water vapor concentration. Within the scope of this work the fluorescence yield for the eight strongest nitrogen emission bands between 300 nm and 400 nm has been measured using electrons from a Sr-90 source with energies between 250 keV and 2000 keV. Measurements have been performed in dry air, pure nitrogen, and a nitrogen-oxygen mixture at pressures ranging from 2 hPa to 990 hPa. Furthermore the influence of water vapor has been studied. A new approach for the parametrization of the fluorescence yield was used to analyze the data, leading to a consistent description of the fluorescence yield with a minimal set of parameters. The resulting absolute accuracies for the single nitrogen bands are in the order of 15%. In the investigated energy range, the fluorescence yield proved to be independent of

Fluorescence molecular tomography (FMT) is a promising modality for non-invasive imaging of internal fluorescence agents in biological tissues especially in small animal models, with applications in diagnosis, therapy, and drug design. In this paper, we present a new fluorescent reconstruction algorithm that combines time-resolved fluorescence imaging data with photon-counting micro-CT (PCMCT) images to estimate the quantum yield and lifetime of fluorescent markers in a mouse model. By incorporating PCMCT images, a permissible region of interest of fluorescence yield and lifetime can be roughly estimated as prior knowledge, reducing the number of unknown variables in the inverse problem and improving image reconstruction stability. Our numerical simulation results demonstrate the accuracy and stability of this method in the presence of data noise, with an average relative error of 18% in fluorescent yield and lifetime reconstruction.

Extensive air showers initiate the fluorescence emissions from nitrogen molecules in air. The UV-light is emitted isotropically and can be used for observing the longitudinal development of extensive air showers in the atmosphere over tenth of kilometers. This measurement technique is well-established since it is exploited for many decades by several cosmic ray experiments. However, a fundamental aspect of the air shower analyses is the description of the fluorescence emission in dependence on varying atmospheric conditions. Different fluorescence yields affect directly the energy scaling of air shower reconstruction. In order to explore the various details of the nitrogen fluorescence emission in air, a few experimental groups have been performing dedicated measurements over the last decade. Most of the measurements are now finished. These experimental groups have been discussing their techniques and results in a series of Air Fluorescence Workshops commenced in 2002. At the 8$^{\rm{th}}$ Air Fluorescence Workshop 2011, it was suggested to develop a common way of describing the nitrogen fluorescence for application to air shower observations. Here, first analyses for a common trea

We predict intensities of lines of CII, NI, NII, OI and OII and compare them with a deep spectroscopic survey of IC 418 to test the effect of excitation of nebular emission lines by continuum fluorescence of starlight. Our calculations use a nebular model and a synthetic spectrum of its central star to take into account excitation of the lines by continuum fluorescence and recombination. The NII spectrum is mostly produced by fluorescence due to the low excitation conditions of the nebula, but many CII and OII lines have more excitation by fluorescence than recombination. In the neutral envelope, the NI permitted lines are excited by fluorescence, and almost all the OI lines are excited by recombination. Electron excitation produces the forbidden optical lines of OI, but continuum fluorescence excites most of the NI forbidden line intensities. Lines excited by fluorescence of light below the Lyman limit thus suggest a new diagnostic to explore the photodissociation region of a nebula.

First discovered by Ernest Abbe in 1873, the resolution limit of a far-field microscope is considered determined by the numerical aperture and wavelength of light, approximately $λ$/2NA. With the advent of modern fluorescence microscopy and nanoscopy methods over the last century, it is recognized that Abbe's resolution definition alone could not solely characterize the resolving power of the microscope system. To determine the practical resolution of a fluorescence microscope, photon noise remains one essential factor yet to be incorporated in a statistics-based theoretical framework. Techniques such as confocal allow trading photon noise in gaining its resolution limit, which may increase or worsen the resolvability towards fluorescently tagged targets. Proposed as a theoretical measure of fluorescence microscopes' resolving power with finite photons, we quantify the resolvability of periodic structures in fluorescence microscopy systems considering both the diffraction limit and photon statistics. Using the Cramer-Rao Lower Bound of a parametric target, the resulting precision lower bound establishes a practical measure of the theoretical resolving power for various modern fluor

Fluorescence microscopy is a powerful tool for imaging biological samples with molecular specificity. In contrast, phase microscopy provides label-free measurement of the sample's refractive index (RI), which is an intrinsic optical property that quantitatively relates to cell morphology, mass, and stiffness. Conventional imaging techniques measure either the labeled fluorescence (functional) information or the label-free RI (structural) information, though it may be valuable to have both. For example, biological tissues have heterogeneous RI distributions, causing sample-induced scattering that degrades the fluorescence image quality. When both fluorescence and 3D RI are measured, one can use the RI information to digitally correct multiple-scattering effects in the fluorescence image. Here, we develop a new computational multi-modal imaging method based on epi-mode microscopy that reconstructs both 3D fluorescence and 3D RI from a single dataset. We acquire dozens of fluorescence images, each 'illuminated' by a single fluorophore, then solve an inverse problem with a multiple-scattering forward model. We experimentally demonstrate our method for epi-mode 3D RI imaging and digital

A scanning tunneling microscope is used to study the fluorescence of a model charged molecule (quinacridone) adsorbed on a sodium chloride (NaCl)-covered metallic sample. Fluorescence from the neutral and positively charged species is reported and imaged using hyper-resolved fluorescence microscopy. A many-body excitation model is established based on a detailed analysis of voltage, current and spatial dependencies of the fluorescence and electron transport features. This model reveals that quinacridone adopts a large palette of charge states, transient or not, depending on the voltage used and the nature of the underlying substrate. This model has a universal character and explains the electronic and fluorescence properties of many other molecules adsorbed on thin insulators.

In this paper, we propose an x-ray fluorescence imaging system for elemental analysis. The key idea is what we call "x-ray fluorescence sectioning". Specifically, a slit collimator in front of an x-ray tube is used to shape x-rays into a fan-beam to illuminate a planar section of an object. Then, relevant elements such as gold nanoparticles on the fan-beam plane are excited to generate x-ray fluorescence signals. One or more 2D spectral detectors are placed to face the fan-beam plane and directly measure x-ray fluorescence data. Detector elements are so collimated that each element only sees a unique area element on the fan-beam plane and records the x-ray fluorescence signal accordingly. The measured 2D x-ray fluorescence data can be refined in reference to the attenuation characteristics of the object and the divergence of the beam for accurate elemental mapping. This x-ray fluorescence sectioning system promises fast fluorescence tomographic imaging without a complex inverse procedure. The design can be adapted in various ways, such as with the use of a larger detector size to improve the signal to noise ratio. In this case, the detector(s) can be shifted multiple times for imag

The absolute value of the air-fluorescence yield is a key parameter for the energy reconstruction of extensive air showers registered by fluorescence telescopes. In previous publications, we reported a detailed Monte Carlo simulation of the air-fluorescence generation that allowed the theoretical evaluation of this parameter. This simulation has been upgraded in the present work. As a result, we determined an updated absolute value of the fluorescence yield of 7.9+-2.0 ph/MeV for the band at 337 nm in dry air at 800 hPa and 293 K, in agreement with experimental values. We have also performed a critical analysis of available absolute measurements of the fluorescence yield with the assistance of our simulation. Corrections have been applied to some measurements to account for a bias in the evaluation of the energy deposition. Possible effects of other experimental aspects have also been discussed. From this analysis, we determined an average fluorescence yield of 7.04+-0.24 ph/MeV at the above conditions.

Fluorescence microscopy has enabled a dramatic development in modern biology. Due to its inherently weak signal, fluorescence microscopy is not only much noisier than photography, but also presented with Poisson-Gaussian noise where Poisson noise, or shot noise, is the dominating noise source. To get clean fluorescence microscopy images, it is highly desirable to have effective denoising algorithms and datasets that are specifically designed to denoise fluorescence microscopy images. While such algorithms exist, no such datasets are available. In this paper, we fill this gap by constructing a dataset - the Fluorescence Microscopy Denoising (FMD) dataset - that is dedicated to Poisson-Gaussian denoising. The dataset consists of 12,000 real fluorescence microscopy images obtained with commercial confocal, two-photon, and wide-field microscopes and representative biological samples such as cells, zebrafish, and mouse brain tissues. We use image averaging to effectively obtain ground truth images and 60,000 noisy images with different noise levels. We use this dataset to benchmark 10 representative denoising algorithms and find that deep learning methods have the best performance. To o

We experimentally investigate the resonance fluorescence spectrum of single 171Yb and 172Yb ions which are laser cooled to the Lamb-Dicke regime in a radiofrequency trap. While the fluorescence scattering of 172Yb is continuous, the 171Yb fluorescence is interrupted by quantum jumps because a nonvanishing rate of spontaneous transitions leads to electron shelving in the metastable hyperfine sublevel 2D3/2(F=2). The average duration of the resulting dark periods can be varied by changing the intensity of a repumping laser field. Optical heterodyne detection is employed to analyze the fluorescence spectrum near the Rayleigh elastic scattering peak. It is found that the stochastic modulation of the fluorescence emission by quantum jumps gives rise to a Lorentzian component in the fluorescence spectrum, and that the linewidth of this component varies according to the average duration of the dark fluorescence periods. The experimental observations are in quantitative agreement with theoretical predictions.