Changes In Fluorescence Lifetime Research Articles

Fluorescence lifetime imaging of human pancreatic lipase activity using a novel probe for early diagnosis of severe acute pancreatitis

Severe Acute Pancreatitis, a serious condition caused by factors such as gallstones and chronic excessive alcohol consumption, with a very high mortality rate. Human pancreatic lipase (hPL) is a key digestive enzyme and abnormal activity levels of this enzyme are important indicators for diagnosing and monitoring pancreatic diseases. A fluorescent probe, LPP, has been developed to monitor the activity of hPL, especially in cases of SAP. The probe is based on cyanine isoindole derivatives, in vitro experiments confirmed the high specificity and sensitivity of the probe, with a detection limit of 0.012 U/mL, reactions completed within 10 min, and effective monitoring of pancreatic lipase activity in various biological samples. The stability and low cytotoxicity of LPP make it suitable for clinical applications, providing new tools and perspectives for the research and treatment of pancreatic diseases and related metabolic abnormalities. In addition, the change in fluorescence lifetime after the reaction of the probe with lipase allows for fluorescence lifetime imaging (FLIM), effectively monitoring the dynamic changes of hPL and enabling early diagnosis and monitoring of pancreatitis. This research not only enhances the understanding of pancreatic lipase activity detection but also has the potential to improve the diagnostics and treatment of pancreatitis.

Multiplexed In Vivo Imaging with Fluorescence Lifetime-Modulating Tags.

Fluorescence lifetime imaging microscopy (FLIM) opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives is described. It is shown that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime-modulating tags enable multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish larvae. The combination of fluorescence lifetime multiplexing with spectral multiplexing allows to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy

Fluorescence microscopy is limited by photoconversion due to continuous illumination, which results in not only photobleaching but also conversion of fluorescent molecules into species of different spectral properties through photoblueing. Here, we determined different fluorescence parameters of photoconverted products for various fluorophores under standard confocal and stimulated emission depletion (STED) microscopy conditions. We observed changes in both fluorescence spectra and lifetimes that can cause artifacts in quantitative measurements, which can be avoided by using exchangeable dyes.

Luminescence enhancement of Y2O3 thin films based on LSPR effect of Ag nanolayers

Based on the local surface plasmon resonance effect of Ag nanolayer, the luminescence enhancement in the visible light range of multilayer thin film has been investigated combined with metal/spacer molecule/photoluminescence material structure. The luminescence enhancement effect of multilayer thin films can be improved by adjusting the spin-coating number of Ag nanolayer and SiO2 spacer in the multilayer structure. At the same time, the mechanism of enhanced luminescence has been analyzed according to the change in fluorescence lifetime. The effect of the coupling between Ag nanolayer and luminescence layer on the radiative transition rate and fluorescence quantum yield have been discussed. The up-conversion luminescence intensity of the noble metal-modified multilayer thin film can be further improved by combining the multilayer structure with sensitized layer.

A Toxicogenic Interaction between Intracellular Amyloid-β and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid β (Aβ) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aβ oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aβ-ApoE interaction suppresses these changes and concomitantly reduces Aβ toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aβ oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Fast and slow: Recording neuromodulator dynamics across both transient and chronic time scales.

Neuromodulators transform animal behaviors. Recent research has demonstrated the importance of both sustained and transient change in neuromodulators, likely due to tonic and phasic neuromodulator release. However, no method could simultaneously record both types of dynamics. Fluorescence lifetime of optical reporters could offer a solution because it allows high temporal resolution and is impervious to sensor expression differences across chronic periods. Nevertheless, no fluorescence lifetime change across the entire classes of neuromodulator sensors was previously known. Unexpectedly, we find that several intensity-based neuromodulator sensors also exhibit fluorescence lifetime responses. Furthermore, we show that lifetime measures in vivo neuromodulator dynamics both with high temporal resolution and with consistency across animals and time. Thus, we report a method that can simultaneously measure neuromodulator change over transient and chronic time scales, promising to reveal the roles of multi-time scale neuromodulator dynamics in diseases, in response to therapies, and across development and aging.

Spectroscopic Identification of Charge Transfer of Thiolated Molecules on Gold Nanoparticles via Gold Nanoclusters.

Investigation of charge transfer needs analytical tools that could reveal this phenomenon, and enables understanding of its effect at the molecular level. Here, we show how the combination of using gold nanoclusters (AuNCs) and different spectroscopic techniques could be employed to investigate the charge transfer of thiolated molecules on gold nanoparticles (AuNP@Mol). It was found that the charge transfer effect in the thiolated molecule could be affected by AuNCs, evidenced by the amplification of surface-enhanced Raman scattering (SERS) signal of the molecule and changes in fluorescence lifetime of AuNCs. Density functional theory (DFT) calculations further revealed that AuNCs could amplify the charge transfer process at the molecular level by pumping electrons to the surface of AuNPs. Finite element method (FEM) simulations also showed that the electromagnetic enhancement mechanism along with chemical enhancement determines the SERS improvement in the thiolated molecule. This study provides a mechanistic insight into the investigation of charge transfer at the molecular level between organic and inorganic compounds, which is of great importance in designing new nanocomposite systems. Additionally, this work demonstrates the potential of SERS as a powerful analytical tool that could be used in nanochemistry, material science, energy, and biomedical fields.

Three-Four Photon Transition Mn(II) Complex Monitoring Lysosome-Related ATP in Real Time via Fluorescence Lifetime Imaging.

Currently, in situ monitoring of the adenosine triphosphate (ATP) level in lysosomes is critical to understand their involvement in various biological processes, but it remains difficult due to the interferences of limited targeting and low resolution of fluorescent probes. Herein, we report a classic Mn(II) probe (FX2-MnCl2) with near-infrared (NIR) nonlinear (NLO) properties, accompanied by three-four photon transition and fivefold fluorescence enhancement in the presence of ATP. FX2-MnCl2 combines with ATP through dual recognition sites of diethoxy and manganese ions to reflect slightly fluorescence lifetime change. Through the synergy of multiphoton fluorescence imaging (MP-FI) and multiphoton fluorescence lifetime imaging microscopy (MP-FLIM), it is further demonstrated that FX2-MnCl2 displays lysosome-specific targeting behavior, which can monitor lysosome-related ATP migration under NIR laser light. This work provides a novel multiphoton transformation fluorescence complex, which might be a potential candidate as a simple and straightforward biomarker of lysosome ATP in vitro for clinical diagnosis.

Fibre-based fluorescence-lifetime imaging microscopy: a real-time biopsy guidance tool for suspected lung cancer.

Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P<0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer.

Phasor-FLIM analysis of cellulose paper ageing mechanism with carbotrace 680 dye

Ageing of paper is a complex process of great relevance for application purposes because of its widespread use as support for information storage in books and documents, and as common low-cost and green packaging material, to name a few. A key factor in paper ageing is the oxidation of cellulose, a macromolecule of natural origin that constitutes the main chemical component of paper. Such a complex process results in changes in the cellulose polymeric chains in chemical and structural properties. The scope of this work is to explore the effects of oxidation of cellulose as one of the principal mechanisms of ageing of paper using a fluorescence-based approach. To this aim, fluorescence-lifetime imaging microscopy (FLIM) measurements on pure cellulose samples stained using Carbotrace 680 dye were performed, and data were analyzed by phasor approach. The comparison with results from conventional techniques allowed to map paper microstructure as a function of the sample oxidation degree correlating the fluorescence-lifetime changes to cellulose oxidation. A two-step oxidation kinetics that produced specific modification in paper organization was highlighted indicating that FLIM measurements using Carbotrace 680 dye may provide a simple tool to obtain information on the oxidation process also adding spatial information at sub-micrometric scale.

Evaluating Riboglow-FLIM probes for RNA sensing.

We recently developed Riboglow-FLIM, where we genetically tag and track RNA molecules in live cells through measuring the fluorescence lifetime of a small molecule probe that binds the RNA tag. Here, we systematically and quantitatively evaluated key elements of Riboglow-FLIM that may serve as the foundation for Riboglow-FLIM applications and further tool development efforts. Our investigation focused on measuring changes in fluorescence lifetime of representative Riboglow-FLIM probes with different linkers and fluorophores in different environments. In vitro measurements revealed distinct lifetime differences among the probe variants as a result of different linker designs and fluorophore selections. To expand on the platform's versatility, probes in a wide variety of mammalian cell types were examined using fluorescence lifetime imaging microscopy (FLIM), and possible effects on cell physiology were evaluated by metabolomics. The results demonstrated that variations in lifetime were dependent on both probe and cell type. Interestingly, distinct differences in lifetime values were observed between cell lines, while no overall change in cell health was measured. These findings underscore the importance of probe selection and cellular environment when employing Riboglow-FLIM for RNA detection, serving as a foundation for future tool development and applications across diverse fields and biological systems.

Observing bioorthogonal macrocyclizations in the nuclear envelope of live cells using on/on fluorescence lifetime microscopy.

The reactive partnership between azides and strained alkynes is at the forefront of bioorthogonal reactions, with their in situ cellular studies often achieved through the use of off to on fluorophores with fluorescence microscopy. In this work, the first demonstration of a bioorthogonal, macrocycle-forming reaction occurring within the nuclear envelope of live cells has been accomplished, utilising on/on fluorescence lifetime imaging microscopy for real-time continuous observation of the transformation. The fluorescent, macrocyclic BF2 azadipyrromethene was accessible through a double 1,3-dipolar cycloaddition within minutes, between a precursor bis-azido substituted fluorophore and Sondheimer diyne in water or organic solvents. Photophysical properties of both the starting bis-azide BF2 azadipyrromethene and the fluorescent macrocyclic products were obtained, with near identical emission wavelengths and intensities, but different lifetimes. In a novel approach, the progress of the live-cell bioorthogonal macrocyclization was successfully tracked through a fluorescence lifetime change of 0.6 ns from starting material to products, with reaction completion achieved within 45 min. The continuous monitoring and imaging of this bioorthogonal transformation in the nuclear membrane and invaginations, of two different cancer cell lines, has been demonstrated using a combination of fluorescence intensity and lifetime imaging with phasor plot analysis. As there is a discernible difference in fluorescence lifetimes between starting material and products, this approach removes the necessity for off-to-on fluorogenic probes when preparing for bioorthogonal cell-imaging and microscopy.

PDT-Induced Variations of Radachlorin Fluorescence Lifetime in Living Cells In Vitro

Variations in the fluorescence lifetimes of Radachlorin photosensitizers in HeLa and A549 cells, caused by photodynamic treatment, were studied using fluorescence lifetime imaging microscopy (FLIM). An analysis of FLIM images of the cells demonstrated a substantial decrease in the mean Radachlorin fluorescence lifetime and intensity as a result of UV irradiation of the photosensitized cells at different doses, with higher doses causing a more pronounced decrease in the mean fluorescence lifetime in cells. The post-treatment decrease in Radachlorin fluorescence intensity was accompanied by the appearance of an additional rapidly decaying fluorescence component and a nonlinear decrease in the weighted fluorescence lifetime obtained from double-exponential fits of time-resolved fluorescence signals. Experiments performed in the aqueous solutions of the photosensitizer revealed similar irreversible changes in the Radachlorin fluorescence lifetime and intensity. Therefore, the observed phenomena occurred most likely due to the photodegradation of the photosensitizer molecules and can be applied for dosimetry and monitoring of irradiation doses in different areas of malignant tissues in the course of photodynamic treatment.

Steady-State and Time-Resolved Fluorescence Study of Selected Tryptophan-Containing Peptides in an AOT Reverse Micelle Environment.

The aim of this study was to demonstrate the utility of time-resolved fluorescence spectroscopy in the detection of subtle changes in the local microenvironment of a tryptophan chromophore in a confined and crowded medium of AOT reverse micelles, which mimic biological membranes and cell compartmentalization. For this purpose, fluorescence properties of L-tryptophan and several newly synthesized tryptophan-containing peptides in buffer and in an AOT reverse micelle medium were determined. It was shown that insertion of tryptophan and its short di- and tripeptides inside micelles led to evident changes in both the steady-state emission spectra and in fluorescence decay kinetics. The observed differences in spectral characteristics, such as a blue shift in the emission maxima, changes in the average fluorescence lifetime, and the appearance of environmental-dependent fluorescent species, showed the utility of time-resolved fluorescence spectroscopy as a sensitive tool for detecting subtle conformational modifications in tryptophan and its peptides induced by changes in polarity, viscosity, and specific interactions between chromophores and water molecules/polar groups/ions that occur inside reverse micelles.

Spectroscopy evidence of interaction of 1,5 disubstituted piperidino-amido anthraquinone derivative with human telomeric G-quadruplex DNA: Basis of anticancer action

The binding of ligands to G-quadruplex (G4) structures at the ends of human telomeric DNA induces thermal stabilization, which interferes with telomere maintenance by disrupting the association of telomeres with the telomerase enzyme—an important marker for cancer. Understanding the binding mode of the ligand-G quadruplex DNA complex is imperative for evaluating the relative efficacy and specificity of their interaction. We focused on the interaction of 1,5-Bis(3-piperidino propionamido) anthracene-9,10-dione with human telomeric DNA sequences using surface plasmon resonance, absorbance, fluorescence (steady-state and lifetime), and circular dichroism spectroscopy techniques. The ligand binding with HTel22 (Kb = 8.4 × 105 M−1) induced cell toxicity with an IC50 value of ∼ 8.64 µM in MCF-7 cancer cell lines. Significant hypochromism (59 %), fluorescence quenching (97 %), no change in fluorescence lifetime and absence of induced Circular Dichroism (CD) band upon addition of G4 DNA to ligand, suggest a groove/external binding mode. CD spectral changes reflect rearrangement in parallel and antiparallel strands to accommodate the ligand. Docking results reveal specific short contacts of the ligand's side chain, including 9CO, 13N, 14NH, and 11CO, with the grooves of the G4 DNA, without making any contact with the loops, favoring an energetically more favorable conformation. Thermal denaturation profiles, obtained by Differential Scanning Calorimetry and CD, show the stabilization of wHTel26 and HTel22 G4 DNA in K+ and Na+ rich solutions by 21.2 and 17.6 °C, respectively, which may restrict the access of telomerase to telomeres. The findings indicate the potential of modifying anthraquinone substituent groups for therapeutic applications.

Photophysical properties of Radachlorin photosensitizer in solutions of different pH, viscosity and polarity

We present a thorough experimental investigation of fluorescence properties of Radachlorin photosensitizer in solutions of different acidity, viscosity and polarity. Experiments were performed using time-resolved fluorescence lifetime imaging and time-resolved analysis of polarized fluorescence. Variations of solution acidity resulted in considerable changes of Radachlorin fluorescence quantum yield and lifetime in the pH range from 4 to 7, but did not affect the rotational diffusion time, and almost did not change the quantum yield and characteristic times of singlet oxygen phosphorescence. Variations of solution polarity and viscosity were achieved by changing ethanol or methanol fraction in aqueous solution. The decrease of solution polarity resulted in nonlinear rise of Radachlorin fluorescence quantum yield and lifetime up to alcohol concentration of 50%–65%, as well as in considerable rise of singlet oxygen quantum yield and significant changes in characteristic times of its phosphorescence. Variations of solution viscosity resulted in changes of rotational diffusion time of Radachlorin molecules, which appeared to be in perfect correlation with methanol solution viscosity. Good correspondence with ethanol solution viscosity was observed only up to 50% alcohol fraction. Deviations of rotational diffusion time of Radachlorin molecules from direct proportionality with solution viscosity at higher ethanol concentrations were suggested to be due to different solvation conditions. The data obtained can give important reference points for analysis of microenvironment of Radachlorin molecules, their intracellular localization and performance in singlet oxygen generation.

Time-Resolved Fluorescence Spectroscopy of Blood, Plasma and Albumin as a Potential Diagnostic Tool for Acute Inflammation in COVID-19 Pneumonia Patients.

Fluorescence lifetime measurements of blood or plasma offer valuable insights into the microenvironment and molecular interactions of fluorophores, particularly concerning albumin. Neutrophil- and hypoxia-induced oxidative stress in COVID-19 pneumonia patients leads to hyperinflammation, various oxidative modifications of blood proteins, and potential alterations in the fluorescence lifetime of tryptophan-containing proteins, especially albumin. The objective of this study was to investigate the efficacy of time-resolved fluorescence spectroscopy of blood and plasma as a prompt diagnostic tool for the early diagnosis and severity assessment of COVID-19-associated pneumonia. This study examined a cohort of sixty COVID-19 patients with respiratory symptoms. To investigate whether oxidative stress is the underlying cause of the change in fluorescence lifetime, human serum albumin was treated with chloramine T. The time-resolved spectrometer Life Spec II (Edinburgh Instruments Ltd., Livingston, UK), equipped with a sub-nanosecond pulsed 280 nm diode, was used to measure the fluorescence lifetime of blood and plasma. The findings revealed a significant reduction in the fluorescence lifetime of blood (diluted 200 times) and plasma (diluted 20 times) at 360 nm in COVID-19 pneumonia patients compared with their respective values recorded six months post-infection and those of healthy individuals. Significant negative correlations were observed between the mean fluorescence lifetime of blood and plasma at 360 nm and several severity biomarkers and advanced oxidation protein products, while a positive correlation was found with albumin and the albumin-globulin ratio. The time-resolved fluorescence spectroscopy method demonstrates the potential to be used as a preliminary screening technique for identifying patients who are at risk of developing severe complications. Furthermore, the small amount of blood required for the measurements has the potential to enable a rapid fingerstick blood test.

Efficient single-cell oxygen consumption rate characterization based on frequency domain fluorescence lifetime imaging microscopy measurement and microfluidic platform.

Cell metabolism is critical in regulating normal cell functions to maintain energy homeostasis. In order to monitor cell metabolism, the oxygen consumption rate (OCR) of cells has been characterized as an important factor. In conventional cell analysis, the cells are characterized in bulk due to technical limitations. However, the heterogeneity between the cells cannot be identified. Therefore, single-cell analysis has been proposed to reveal cellular functions and their heterogeneity. In this research, an approach integrating a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell OCR characterization in an efficient manner is developed. The microfluidic device provides an efficient platform to trap and isolate single cells in microwells with the buffer saline containing an oxygen-sensitive phosphorescent dye. The oxygen tension variation within the microwells can be efficiently estimated by measuring the fluorescence lifetime change using the FD-FLIM, and the OCR values of the single cells can then be calculated. In the experiments, breast cancer (MCF-7) cells are exploited for the OCR measurement. The results demonstrate the functionality of the developed approach and show the heterogeneity among the cells. The developed approach possesses great potential to advance cellular metabolism studies with single-cell resolution.

LUMINESCENT PROPERTIES OF Nd(III) COMPLEXES WITH ETHY­LENE­DIAMINE-N,N'-DISUCCINIC AND N,N-BIS(PHOSPHONO­METHYL)-2-AMINOPROPIONIC ACIDS

An analysis of the fluorescent characteristics of ethylenediamine-N,N'-disuccinic and N,N-bis(phosphonomethyl)-2-aminopropionic acids was carried out depending on the pH of the solutions. It was established that the change in fluorescence intensity and lifetime is associated with the formation of variously protonated forms of acids in which stable H-cycles are formed with the participation of hydrogen bonds. The energies of the singlet and triplet levels of the ligands were experimentally determined, the values of which are higher than the energy of the radiative level of the Nd(III) ion, which indicates the possibility of intramolecular transfer of the excitation energy to the resonance level of the lanthanide ion. It was established that both homo- and heteronuclear complexes of Nd(III) exhibit 4f-luminescence in the near-IR region. It was found that for phosphorus-containing complexes there is an increase in luminescence intensity and relative quantum yields in comparison with aminocarboxylate analogs. In heterometallic complexes based on aminopolycarboxylic acids, the intramolecular transfer of energy from the excited level of Co(II) to the resonance level of the f-metal leads to sensitization of the 4f-luminescence of the neodymium ion.

Increased intracellular crowding during hyperosmotic stress

Hyperosmotic stress activates in live cells numerous processes and also promotes intracellular protein/RNA aggregation and phase separation. However, the time course and the extent of these changes remain largely uncharacterized. To investigate dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress in live cells, we used fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy (FCS) to quantify changes in the local environment by measuring the fluorescence lifetime and the diffusion of the monomeric enhanced green fluorescent protein (eGFP), respectively. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the acceptor/donor emission ratio using the MMC-sensitive Förster resonance energy transfer sensor (GimRET). This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, eGFP diffusion assessed by FCS indicated higher intracellular viscosity due to increased MMC during hyperosmotic stress. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. Our approach can therefore be used to reveal in live cells short-lived transient states through which MMC builds over time, which could not be observed when measuring changes in other physical properties that occur at slower time scales.