Methods and Applications in Fluorescence - IOPscience

Methods and Applications in Fluorescence - IOPscience
Methods and Applications in Fluorescence
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Methods and Applications in Fluorescence
is an internationally leading journal, which welcomes contributions on the study, application, techniques and instrumentation of fluorescence. There are no page charges.
Submissions are invited for the
Special Issue Featuring Selected Papers from MAF 2025
in Montréal, the 19th edition of the World's largest and longest established
conference series
dedicated to fluorescence.
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2.4
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The following article is
Open access
Photophysics of thermally activated delayed fluorescence molecules
Fernando B Dias
et al
2017
Methods Appl. Fluoresc.
012001
View article
, Photophysics of thermally activated delayed fluorescence molecules
PDF
, Photophysics of thermally activated delayed fluorescence molecules
Thermally activated delayed fluorescence (TADF) has recently emerged as one of the most attractive methods for harvesting triplet states in metal-free organic materials for application in organic light emitting diodes (OLEDs). A large number of TADF molecules have been reported in the literature with the purpose of enhancing the efficiency of OLEDs by converting non-emissive triplet states into emissive singlet states. TADF emitters are able to harvest both singlets and triplet states through fluorescence (prompt and delayed), the latter due to the thermally activated reverse intersystem crossing mechanism that allows up-conversion of low energy triplet states to the emissive singlet level. This allows otherwise pure fluorescent OLEDs to overcome their intrinsic limit of 25% internal quantum efficiency (IQE), which is imposed by the 1:3 singlet–triplet ratio arising from the recombination of charges (electrons and holes). TADF based OLEDS with IQEs close to 100% are now routinely fabricated in the green spectral region. There is also significant progress for blue emitters. However, red emitters still show relatively low efficiencies. Despite the significant progress that has been made in recent years, still significant challenges persist to achieve full understanding of the TADF mechanism and improve the stability of these materials. These questions need to be solved in order to fully implement TADF in OLEDs and expand their application to other areas. To date, TADF has been exploited mainly in the field of OLEDs, but applications in other areas, such as sensing and fluorescence microscopies, are envisaged. In this review, the photophysics of TADF molecules is discussed, summarising current methods to characterise these materials and the current understanding of the TADF mechanism in various molecular systems.
The following article is
Open access
An introduction to optical super-resolution microscopy for the adventurous biologist
J Vangindertael
et al
2018
Methods Appl. Fluoresc.
022003
View article
, An introduction to optical super-resolution microscopy for the adventurous biologist
PDF
, An introduction to optical super-resolution microscopy for the adventurous biologist
Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological
tour de force
to a staple of the biologist’s toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.
The following article is
Open access
Microscale thermophoresis as a powerful growing analytical technique for the investigation of biomolecular interaction and the determination of binding parameters
Sami El Deeb
et al
2022
Methods Appl. Fluoresc.
10
042001
View article
, Microscale thermophoresis as a powerful growing analytical technique for the investigation of biomolecular interaction and the determination of binding parameters
PDF
, Microscale thermophoresis as a powerful growing analytical technique for the investigation of biomolecular interaction and the determination of binding parameters
The
in vitro
panel of technologies to address biomolecular interactions are in play, however microscale thermophoresis is continuously increasing in use to represent a key player in this arena. This review highlights the usefulness of microscale thermophoresis in the determination of molecular and biomolecular affinity interactions. This work reviews the literature from January 2016 to January 2022 about microscale thermophoresis. It gives a summarized overview about both the state-of the art and the development in the field of microscale thermophoresis. The principle of microscale thermophoresis is also described supported with self-created illustrations. Moreover, some recent advances are mentioned that showing application of the technique in investigating biomolecular interactions in different fields. Finally, advantages as well as drawbacks of the technique in comparison with other competing techniques are summarized.
The following article is
Open access
Fluorescence Guided Surgery
Hazel L Stewart and David J S Birch 2021
Methods Appl. Fluoresc.
042002
View article
, Fluorescence Guided Surgery
PDF
, Fluorescence Guided Surgery
Fluorescence guided surgery (FGS) is an imaging technique that allows the surgeon to visualise different structures and types of tissue during a surgical procedure that may not be as visible under white light conditions. Due to the many potential advantages of fluorescence guided surgery compared to more traditional clinical imaging techniques such as its higher contrast and sensitivity, less subjective use, and ease of instrument operation, the research interest in fluorescence guided surgery continues to grow over various key aspects such as fluorescent probe development and surgical system development as well as its potential clinical applications. This review looks to summarise some of the emerging opportunities and developments that have already been made in fluorescence guided surgery in recent years while highlighting its advantages as well as limitations that need to be overcome in order to utilise the full potential of fluorescence within the surgical environment.
The following article is
Open access
Challenges and limitations of molecular resolution fluorescence imaging
Dominic A Helmerich and Markus Sauer 2025
Methods Appl. Fluoresc.
13
043101
View article
, Challenges and limitations of molecular resolution fluorescence imaging
PDF
, Challenges and limitations of molecular resolution fluorescence imaging
Super-resolution microscopy (SRM) has revolutionized fluorescence imaging enabling insights into the molecular organization of cells that were previously unconceivable. Latest developments now allow the visualization of individual molecules with nanometer precision and imaging with molecular resolution. However, translating these achievements to imaging under physiological conditions in cells remains challenging. The higher the spatial resolution is pushed by the development of improved SRM methods the more challenging the problems we are confronted when aiming to use them for sub-10 nm fluorescence imaging in cells. It turns out that most developed SRM methods that demonstrate nanometer resolution cannot be directly implemented for molecular resolution imaging in cells. Particularly, fluorescence labeling, i.e. high-density covalent labeling of the molecules of interest with fluorophores with minimal linkage error represents currently a nearly insurmountable obstacle. In addition, even if high labeling densities can be realized it has to be considered that fluorophores can interact via different energy pathways and thus impede super-resolution imaging in the sub-10 nm range. Here, we describe the boundaries, discuss the challenges we must accept and show strategies to circumvent them and achieve true molecular resolution fluorescence imaging under physiological conditions in cells.
The following article is
Open access
Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
Asha Swamy
et al
2026
Methods Appl. Fluoresc.
14
025005
View article
, Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
PDF
, Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
Lipid accumulation has been implicated in the progression of chronic kidney disease, yet its role in glomerulonephritis (GN) remains poorly characterized. Here we use Nile Red (NR), a solvatochromic fluorophore that stains for lipid droplets (LDs) to examine LD distribution and lipid chemistry in glomerular diseases. Seventy-two kidney biopsy samples, including histologically diagnosed GN subtypes and control nephrectomy tissues, were stained with NR. LDs were quantified using a semi-automated MATLAB-based image analysis pipeline and spectral emission profiles were generated to assess the emission ratio, reflecting differences in lipid composition. We found that GN subtypes exhibit distinct patterns of lipid accumulation and lipid polarity as evaluated by using a NR emission ratio. Our study demonstrates the utility of NR fluorescence spectroscopy in detecting disease-specific lipid alterations in GN. The findings suggest that distinct lipid signatures may serve as pathological indicators, offering potential new diagnostic and mechanistic insights into glomerular disease.
The following article is
Open access
Room temperature luminescence of a triangulenium dye ADOTA in PVA films
Zygmunt Gryczynski
et al
2026
Methods Appl. Fluoresc.
14
025003
View article
, Room temperature luminescence of a triangulenium dye ADOTA in PVA films
PDF
, Room temperature luminescence of a triangulenium dye ADOTA in PVA films
The luminescence properties of the cationic triangulenium dye ADOTA embedded in poly(vinyl alcohol) (PVA) were investigated over a broad range of temperatures. We observed extremely efficient delayed fluorescence (DF) with a lifetime of approximately 160 ms. The spectral characteristics of the DF closely match those of the prompt fluorescence. The temperature-dependent emission intensity of ADOTA’s delayed fluorescence reaches a maximum at about 30 °C. This observation reveals for the first time presence of a triplet state T
in triangulenium dyes. Significant intensity and high anisotropy of ADOTA’s DF in time gated detection format offers access to molecular dynamics from micro to millisecond range, in both spectroscopic and microscopic investigations.
The following article is
Open access
Fluorescence microscopy-based quantitation of GLUT4 translocation
Mara Heckmann
et al
2022
Methods Appl. Fluoresc.
10
022001
View article
, Fluorescence microscopy-based quantitation of GLUT4 translocation
PDF
, Fluorescence microscopy-based quantitation of GLUT4 translocation
Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.
The following article is
Open access
FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
Helen M Wilson
et al
2026
Methods Appl. Fluoresc.
14
025004
View article
, FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
PDF
, FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
Robust and interpretable analysis of fluorescence lifetime imaging microscopy (FLIM) data requires careful assessment of data across biological samples. Due to limitations in sample availability, difference in protein expression, photobleaching, or acquisition time, FLIM datasets are often susceptible to signal variability. This is only exacerbated with large field-of-view FLIM data, such as examining metabolic fluxes across whole tissue slices due to morphology changes. We adapt the FLIM F-value (or figure-of-merit) within our analysis as a statistical metric to capture the confidence in lifetime by comparing variance across fitted parameters, analogous to typical image SNR. In this study, we apply pixelwise and regional analysis of F-values across large-area FLIM datasets to identify image regions with similar confidence levels. Visualization of F-value distribution enables detection of acquisition outliers or poor-quality regions within a large mosaic collection, which can be flagged for reacquisition or removal. This approach enhances the statistical power of downstream biological interpretation by ensuring that only data with quantifiable and stable lifetime information are retained. To our knowledge, this is the first application of F-value mapping as a dataset-wide quality control measure in FLIM.
The following article is
Open access
Fiber-optics based fluorescence detection. Part I: Basic concepts
Bong Lee
et al
2024
Methods Appl. Fluoresc.
12
043001
View article
, Fiber-optics based fluorescence detection. Part I: Basic concepts
PDF
, Fiber-optics based fluorescence detection. Part I: Basic concepts
Continuous in-line detection and process monitoring are essential for industrial, analytical, and biomedical applications. Lightweight, highly flexible, and low-cost fiber optics enable the construction of compact and robust hand­held devices for
in situ
chemical and biological species analysis in both industrial and biomedical
in vitro
in vivo
detection. Despite the broad range of fiber-optic based applications, we lack a good understanding of the parameters that govern the efficiency of light collection or the sensitivity of detection. Consequently, comparing samples of different optical density and/or geometry becomes challenging and can lead to misinterpretation of results; especially when we lack the approaches necessary to correct the detected signal (spectra) for artifacts such as inner-filter effect or scattering. Hence, in this work, we discuss factors affecting the signal detected by the fiber optic in the bare and lens-coupled flat-tipped configurations that lead to signal/spectral distortions. We also present a simple generic model describing the excitation profile and emission collection efficiency that we verify with experimental data. Understanding the principles governing the signal collected by the fiber will provide rationales for correcting the measured emission spectra and recovering the true emission profile of optically dense samples.
The following article is
Open access
Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly
Iryna Lysova
et al
2026
Methods Appl. Fluoresc.
14
025006
View article
, Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly
PDF
, Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly
The human immunodeficiency virus 1 (HIV-1) group specific antigen (Gag) polyprotein, the main structural protein of HIV-1 is sufficient to mimic the late stages of the viral replication cycle. When expressed in cells, Gag binds to RNAs and assembles at the plasma membrane to form virus-like particles (VLPs) with a morphology similar to that of HIV-1 virions. The nucleocapsid (NC) domain of Gag plays a critical role in RNA binding and selective encapsidation of the viral genome. This work focuses on the impact of NC deletion on Gag assembly, VLP formation, and intracellular trafficking. By combining several fluorescence-based quantitative microscopy techniques, we show that in the absence of the NC domain, Gag cytoplasmic oligomerization and formation of initial ribonucleoprotein complexes are impacted, resulting in a significant delay in the kinetics of VLP formation. Interestingly, VLPs formed from the Gag-ΔNC mutant display greater diversity in size and shape. Single particle tracking experiments revealed that VLPs formed at the plasma membrane are immobile, while intracellular VLPs are mostly mobile. For the latter, the motions and diffusion coefficients of VLPs formed by the Gag-ΔNC mutant were highly similar to those of VLPs formed with the wild-type Gag, indicating that the NC domain is not implicated in the intracellular trafficking. These findings illustrate how fluorescence-based microscopy techniques can provide quantitative insights into the role of the NC domain in Gag assembly.
The following article is
Open access
Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
Asha Swamy
et al
2026
Methods Appl. Fluoresc.
14
025005
View article
, Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
PDF
, Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
Lipid accumulation has been implicated in the progression of chronic kidney disease, yet its role in glomerulonephritis (GN) remains poorly characterized. Here we use Nile Red (NR), a solvatochromic fluorophore that stains for lipid droplets (LDs) to examine LD distribution and lipid chemistry in glomerular diseases. Seventy-two kidney biopsy samples, including histologically diagnosed GN subtypes and control nephrectomy tissues, were stained with NR. LDs were quantified using a semi-automated MATLAB-based image analysis pipeline and spectral emission profiles were generated to assess the emission ratio, reflecting differences in lipid composition. We found that GN subtypes exhibit distinct patterns of lipid accumulation and lipid polarity as evaluated by using a NR emission ratio. Our study demonstrates the utility of NR fluorescence spectroscopy in detecting disease-specific lipid alterations in GN. The findings suggest that distinct lipid signatures may serve as pathological indicators, offering potential new diagnostic and mechanistic insights into glomerular disease.
A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristics
Pragya Parmita Konwar
et al
2026
Methods Appl. Fluoresc.
14
022001
View article
, A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristics
PDF
, A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristics
Atmospheric aerosols affect the climate, ecosystems and human well-being. Accurate detection and classification of aerosols, particularly bioaerosols are essential for effective environmental and public health management. This review presents an overview of laser-induced fluorescence (LIF) Spectroscopy as a powerful approach for real-time, non-destructive aerosol analysis. This study outlines the technological progression from foundational systems to modern commercial instruments such as the ultraviolet aerodynamic particle sizer, wideband integrated bioaerosol sensor, BioScout and Rapid-E. These technologies have provided detailed insights into aerosol size, composition and biological content, yet there are challenges in standardization and signal interpretation. By summarizing key findings and innovations, this study highlights the significance of expanding LIF applications in under-represented regions and encourages the development of robust, field-ready systems to advance air quality management and health safeguards.
Smartphones with multispectral imaging for medical testing
Joseph R Lakowicz
et al
2026
Methods Appl. Fluoresc.
14
023101
View article
, Smartphones with multispectral imaging for medical testing
PDF
, Smartphones with multispectral imaging for medical testing
Modern smartphones equipped with integrated red-green-blue (RGB) cameras are widely used for biomedical imaging and diagnostic assays. However, their limited spectral resolution prevents accurate fluorescence imaging and quantitative sensing. Recent advancements in smartphone-based multispectral imaging (MSI), such as multi-channel CMOS sensors and Bragg mirror–based spectral filters, provide direct multi-wavelength detection with enhanced spectral accuracy. The shift from RGB imaging to MSI smartphones significantly improves fluorescence imaging and sensing by allowing precise differentiation of overlapping emission spectra, true-color fluorescence observation, and quantitative assessment of fluorescent biomarkers. These characteristics are important for fluorescence-based diagnostics, point-of-care testing, and novel applications such as contact-lens sensors and fluorescence-guided imaging. Smartphone-based MSI platforms provide compact, user-friendly, and high-resolution devices that enhance the depth and accuracy of fluorescence imaging and optical sensing in biomedical research and clinical applications.
The following article is
Open access
FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
Helen M Wilson
et al
2026
Methods Appl. Fluoresc.
14
025004
View article
, FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
PDF
, FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
Robust and interpretable analysis of fluorescence lifetime imaging microscopy (FLIM) data requires careful assessment of data across biological samples. Due to limitations in sample availability, difference in protein expression, photobleaching, or acquisition time, FLIM datasets are often susceptible to signal variability. This is only exacerbated with large field-of-view FLIM data, such as examining metabolic fluxes across whole tissue slices due to morphology changes. We adapt the FLIM F-value (or figure-of-merit) within our analysis as a statistical metric to capture the confidence in lifetime by comparing variance across fitted parameters, analogous to typical image SNR. In this study, we apply pixelwise and regional analysis of F-values across large-area FLIM datasets to identify image regions with similar confidence levels. Visualization of F-value distribution enables detection of acquisition outliers or poor-quality regions within a large mosaic collection, which can be flagged for reacquisition or removal. This approach enhances the statistical power of downstream biological interpretation by ensuring that only data with quantifiable and stable lifetime information are retained. To our knowledge, this is the first application of F-value mapping as a dataset-wide quality control measure in FLIM.
A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristics
Pragya Parmita Konwar
et al
2026
Methods Appl. Fluoresc.
14
022001
View article
, A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristics
PDF
, A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristics
Atmospheric aerosols affect the climate, ecosystems and human well-being. Accurate detection and classification of aerosols, particularly bioaerosols are essential for effective environmental and public health management. This review presents an overview of laser-induced fluorescence (LIF) Spectroscopy as a powerful approach for real-time, non-destructive aerosol analysis. This study outlines the technological progression from foundational systems to modern commercial instruments such as the ultraviolet aerodynamic particle sizer, wideband integrated bioaerosol sensor, BioScout and Rapid-E. These technologies have provided detailed insights into aerosol size, composition and biological content, yet there are challenges in standardization and signal interpretation. By summarizing key findings and innovations, this study highlights the significance of expanding LIF applications in under-represented regions and encourages the development of robust, field-ready systems to advance air quality management and health safeguards.
The following article is
Open access
Spectral properties of quinine sulfate in PVA films for front-face format emission measurements
Agnieszka Jablonska
et al
2026
Methods Appl. Fluoresc.
14
023001
View article
, Spectral properties of quinine sulfate in PVA films for front-face format emission measurements
PDF
, Spectral properties of quinine sulfate in PVA films for front-face format emission measurements
The luminescence properties of quinine sulfate (QS) embedded in poly(vinyl alcohol) (PVA) film have been studied in detail. Fluorescence excitation and emission spectra were measured using a front-face configuration, and the corresponding intensities were tabulated. A fluorescence quantum yield of 0.51 was determined for QS-doped PVA, using QS dissolved in 1N H
SO
. Both excitation and emission anisotropy measurements indicated effective immobilization of QS molecules within the polymer matrix. The fluorescence lifetime of QS-doped PVA was measured using a picosecond-pulsed 375 nm laser diode and analyzed with both exponential and Lorentzian distribution models. The broad lifetime distribution suggests a complex interaction between QS molecules and the polymer matrix. The QS-doped PVA films are well suited for measurements involving solid materials and front-face geometries.
The following article is
Open access
Advances in ultraviolet microscopy
M McFarlane and G McConnell 2025
Methods Appl. Fluoresc.
13
042001
View article
, Advances in ultraviolet microscopy
PDF
, Advances in ultraviolet microscopy
Ultraviolet (UV) microscopy is a powerful imaging modality that harnesses the shorter wavelengths of UV light to achieve high-resolution imaging and probe molecular-level chemical and structural properties of biological and biomedical specimens, often without the need for extrinsic labelling. Innovations in technologies such as low-cost illuminators, detectors, and new ways of preparing specimens for imaging have led to a better understanding of complex biological systems. Here we review the latest advances and trends in UV microscopy for applications in the life sciences, including histology, cell biology and haemotology. By examining these developments, we highlight the evolving potential of UV and we conclude by considering the future of this longstanding technique.
The following article is
Open access
Fiber-optics based fluorescence detection. Part I: Basic concepts
Bong Lee
et al
2024
Methods Appl. Fluoresc.
12
043001
View article
, Fiber-optics based fluorescence detection. Part I: Basic concepts
PDF
, Fiber-optics based fluorescence detection. Part I: Basic concepts
Continuous in-line detection and process monitoring are essential for industrial, analytical, and biomedical applications. Lightweight, highly flexible, and low-cost fiber optics enable the construction of compact and robust hand­held devices for
in situ
chemical and biological species analysis in both industrial and biomedical
in vitro
in vivo
detection. Despite the broad range of fiber-optic based applications, we lack a good understanding of the parameters that govern the efficiency of light collection or the sensitivity of detection. Consequently, comparing samples of different optical density and/or geometry becomes challenging and can lead to misinterpretation of results; especially when we lack the approaches necessary to correct the detected signal (spectra) for artifacts such as inner-filter effect or scattering. Hence, in this work, we discuss factors affecting the signal detected by the fiber optic in the bare and lens-coupled flat-tipped configurations that lead to signal/spectral distortions. We also present a simple generic model describing the excitation profile and emission collection efficiency that we verify with experimental data. Understanding the principles governing the signal collected by the fiber will provide rationales for correcting the measured emission spectra and recovering the true emission profile of optically dense samples.
Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research
Lida Aeindartehran
et al
2024
Methods Appl. Fluoresc.
12
032002
View article
, Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research
PDF
, Fluorescence in depth: integration of spectroscopy and imaging with Raman, IR, and CD for advanced research
Fluorescence spectroscopy serves as a vital technique for studying the interaction between light and fluorescent molecules. It encompasses a range of methods, each presenting unique advantages and applications. This technique finds utility in various chemical studies. This review discusses Fluorescence spectroscopy, its branches such as Time-Resolved Fluorescence Spectroscopy (TRFS) and Fluorescence Lifetime Imaging Microscopy (FLIM), and their integration with other spectroscopic methods, including Raman, Infrared (IR), and Circular Dichroism (CD) spectroscopies. By delving into these methods, we aim to provide a comprehensive understanding of the capabilities and significance of fluorescence spectroscopy in scientific research, highlighting its diverse applications and the enhanced understanding it brings when combined with other spectroscopic methods. This review looks at each technique's unique features and applications. It discusses the prospects of their combined use in advancing scientific understanding and applications across various domains.
The following article is
Open access
Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog
Thaysen et al
View accepted manuscript
, Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog
PDF
, Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog
Ergosterol is the main sterol in yeast and an important lipid constituent of the yeast plasma membrane (PM). Methods for analysis of ergosterol trafficking between PM and subcellular compartments often rely on fluorescence microscopy, but existing sterol probes either mimic ergosterol poorly or have inconvenient fluorescence properties. Here, we present a novel intrinsically fluorescent probe that differs from ergosterol only by having a 3'-keto group and two additional conjugated double bonds in the ring system. We show that this analog, named Erg-Tetraene, can order fatty acyl chains of phospholipids and partitions partially into the liquidordered phase in model membranes containing cholesterol. The Erg-Tetraene has a red-shifted emission and a much stronger two-photon absorption than the widely used analog dehydroergosterol, allowing for its convenient imaging on commercial microscope systems. Using multi-color confocal and two-photon microscopy, we show that uptake of Erg-Tetraene into yeast depends on the sterol transporters Aus1/Pdr11 and is followed by rapid transport to the vacuole and to lipid droplets. Together, we present a novel analogue of ergosterol with improved fluorescence properties for sterol trafficking studies in yeast and other model organisms.
The following article is
Open access
A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development
Lopez et al
View accepted manuscript
, A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development
PDF
, A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development
The increasing demand for monoclonal antibodies (mAbs) as therapeutic agents highlights the ever-growing necessity of optimizing sample-selection procedures; the rate-limiting step of therapy development. One such mAb property test is viscosity, which affects syringeability, concentration dose, and patient experience. Current viscometry methods, such as cone and plate rheometry, although accurate, are limited by low throughput, high sample volume requirements, and lack of automation, driving up the cost and time of mAb development. This paper introduces an innovative imaging-based viscometry application that significantly mitigates these issues. By integrating a wide-field camera with a fluorescence microscope and Python-based Single Particle Tracking (SPT) software, this approach allows for rapid, low-volume (down to 2 μL) viscosity measurements of mAb solutions. Using 200 nm yellow-green fluorescent polystyrene beads as tracers, the system ensures accurate macroviscosity assessments of protein solutions and the capability for high-throughput analysis. The platform's precision and sensitivity were validated using Bovine Serum Albumin (BSA) and viscosity standard solutions, followed by a single-blinded study using Immunoglobulin G1 and G2 (IgG1/IgG2) solutions of varying concentrations (≤150 mg/mL) and viscosities (2-31 centipoise). When compared to the unblinded values, the SPT blinded sample analysis resulted in a linear fit of R² = 0.97 and an average error of 2.1 cP. Our findings suggest that this novel platform can substantially streamline and enhance the mAb development process, offering a feasible solution to one of the industry's pressing challenges.
A novel carbon dot-based fluorescence/UV dual-mode sensing method for the detection of organophosphorus pesticides
Luo et al
View accepted manuscript
, A novel carbon dot-based fluorescence/UV dual-mode sensing method for the detection of organophosphorus pesticides
PDF
, A novel carbon dot-based fluorescence/UV dual-mode sensing method for the detection of organophosphorus pesticides
This study presents a fluorescence-colorimetric dual-mode method for the detection of organophosphorus pesticides (OPPs). The method exploits the fact that products of organophosphorus pesticides oxidized by potassium persulfate can both quench the fluorescence of carbon dots and induce color changes. Using Dimethoate (Dim) as the model analyte, the linear detection ranges for the fluorescence and colorimetric modes were 0.05 ~ 4.6 μg/mL and 0.05 ~ 3.0 μg/mL, respectively, with limits of detection (LODs) of 0.027 μg/mL and 0.0109 μg/mL. The synergistic dual-mode response significantly enhances the reliability of the measurements. In spiked recovery tests using actual water samples, both detection modes demonstrated high recovery rates, confirming that the method maintains excellent accuracy and precision even in complex matrices. The dual-mode sensing probe developed in this study features a straightforward preparation process and achieves efficient, sensitive, and reliable detection of multiple OPPs, offering a promising analytical tool for monitoring pesticide residues.
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The following article is
Open access
Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly
Iryna Lysova
et al
2026
Methods Appl. Fluoresc.
14
025006
View article
, Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly
PDF
, Quantitative fluorescence imaging of the impact of the nucleocapsid domain deletion on the dynamics of HIV-1 Gag assembly
The human immunodeficiency virus 1 (HIV-1) group specific antigen (Gag) polyprotein, the main structural protein of HIV-1 is sufficient to mimic the late stages of the viral replication cycle. When expressed in cells, Gag binds to RNAs and assembles at the plasma membrane to form virus-like particles (VLPs) with a morphology similar to that of HIV-1 virions. The nucleocapsid (NC) domain of Gag plays a critical role in RNA binding and selective encapsidation of the viral genome. This work focuses on the impact of NC deletion on Gag assembly, VLP formation, and intracellular trafficking. By combining several fluorescence-based quantitative microscopy techniques, we show that in the absence of the NC domain, Gag cytoplasmic oligomerization and formation of initial ribonucleoprotein complexes are impacted, resulting in a significant delay in the kinetics of VLP formation. Interestingly, VLPs formed from the Gag-ΔNC mutant display greater diversity in size and shape. Single particle tracking experiments revealed that VLPs formed at the plasma membrane are immobile, while intracellular VLPs are mostly mobile. For the latter, the motions and diffusion coefficients of VLPs formed by the Gag-ΔNC mutant were highly similar to those of VLPs formed with the wild-type Gag, indicating that the NC domain is not implicated in the intracellular trafficking. These findings illustrate how fluorescence-based microscopy techniques can provide quantitative insights into the role of the NC domain in Gag assembly.
The following article is
Open access
Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog
Katja Thaysen
et al
2026
Methods Appl. Fluoresc.
View article
, Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog
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, Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog
Ergosterol is the main sterol in yeast and an important lipid constituent of the yeast plasma membrane (PM). Methods for analysis of ergosterol trafficking between PM and subcellular compartments often rely on fluorescence microscopy, but existing sterol probes either mimic ergosterol poorly or have inconvenient fluorescence properties. Here, we present a novel intrinsically fluorescent probe that differs from ergosterol only by having a 3'-keto group and two additional conjugated double bonds in the ring system. We show that this analog, named Erg-Tetraene, can order fatty acyl chains of phospholipids and partitions partially into the liquidordered phase in model membranes containing cholesterol. The Erg-Tetraene has a red-shifted emission and a much stronger two-photon absorption than the widely used analog dehydroergosterol, allowing for its convenient imaging on commercial microscope systems. Using multi-color confocal and two-photon microscopy, we show that uptake of Erg-Tetraene into yeast depends on the sterol transporters Aus1/Pdr11 and is followed by rapid transport to the vacuole and to lipid droplets. Together, we present a novel analogue of ergosterol with improved fluorescence properties for sterol trafficking studies in yeast and other model organisms.
The following article is
Open access
A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development
Karen L Lopez
et al
2026
Methods Appl. Fluoresc.
View article
, A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development
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, A novel imaging-based multi-sample viscometry application for advancing monoclonal antibody development
The increasing demand for monoclonal antibodies (mAbs) as therapeutic agents highlights the ever-growing necessity of optimizing sample-selection procedures; the rate-limiting step of therapy development. One such mAb property test is viscosity, which affects syringeability, concentration dose, and patient experience. Current viscometry methods, such as cone and plate rheometry, although accurate, are limited by low throughput, high sample volume requirements, and lack of automation, driving up the cost and time of mAb development. This paper introduces an innovative imaging-based viscometry application that significantly mitigates these issues. By integrating a wide-field camera with a fluorescence microscope and Python-based Single Particle Tracking (SPT) software, this approach allows for rapid, low-volume (down to 2 μL) viscosity measurements of mAb solutions. Using 200 nm yellow-green fluorescent polystyrene beads as tracers, the system ensures accurate macroviscosity assessments of protein solutions and the capability for high-throughput analysis. The platform's precision and sensitivity were validated using Bovine Serum Albumin (BSA) and viscosity standard solutions, followed by a single-blinded study using Immunoglobulin G1 and G2 (IgG1/IgG2) solutions of varying concentrations (≤150 mg/mL) and viscosities (2-31 centipoise). When compared to the unblinded values, the SPT blinded sample analysis resulted in a linear fit of R² = 0.97 and an average error of 2.1 cP. Our findings suggest that this novel platform can substantially streamline and enhance the mAb development process, offering a feasible solution to one of the industry's pressing challenges.
The following article is
Open access
Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
Asha Swamy
et al
2026
Methods Appl. Fluoresc.
14
025005
View article
, Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
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, Nile red spectroscopy to evaluate disease-specific lipid alterations in glomerular kidney disease
Lipid accumulation has been implicated in the progression of chronic kidney disease, yet its role in glomerulonephritis (GN) remains poorly characterized. Here we use Nile Red (NR), a solvatochromic fluorophore that stains for lipid droplets (LDs) to examine LD distribution and lipid chemistry in glomerular diseases. Seventy-two kidney biopsy samples, including histologically diagnosed GN subtypes and control nephrectomy tissues, were stained with NR. LDs were quantified using a semi-automated MATLAB-based image analysis pipeline and spectral emission profiles were generated to assess the emission ratio, reflecting differences in lipid composition. We found that GN subtypes exhibit distinct patterns of lipid accumulation and lipid polarity as evaluated by using a NR emission ratio. Our study demonstrates the utility of NR fluorescence spectroscopy in detecting disease-specific lipid alterations in GN. The findings suggest that distinct lipid signatures may serve as pathological indicators, offering potential new diagnostic and mechanistic insights into glomerular disease.
The following article is
Open access
FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
Helen M Wilson
et al
2026
Methods Appl. Fluoresc.
14
025004
View article
, FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
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, FLIM quality metric visualization as a means to validate consistency across large-area non-homogeneous FLIM datasets
Robust and interpretable analysis of fluorescence lifetime imaging microscopy (FLIM) data requires careful assessment of data across biological samples. Due to limitations in sample availability, difference in protein expression, photobleaching, or acquisition time, FLIM datasets are often susceptible to signal variability. This is only exacerbated with large field-of-view FLIM data, such as examining metabolic fluxes across whole tissue slices due to morphology changes. We adapt the FLIM F-value (or figure-of-merit) within our analysis as a statistical metric to capture the confidence in lifetime by comparing variance across fitted parameters, analogous to typical image SNR. In this study, we apply pixelwise and regional analysis of F-values across large-area FLIM datasets to identify image regions with similar confidence levels. Visualization of F-value distribution enables detection of acquisition outliers or poor-quality regions within a large mosaic collection, which can be flagged for reacquisition or removal. This approach enhances the statistical power of downstream biological interpretation by ensuring that only data with quantifiable and stable lifetime information are retained. To our knowledge, this is the first application of F-value mapping as a dataset-wide quality control measure in FLIM.
The following article is
Open access
Normalization is essential for accurate intraoperative perfusion assessment using near-infrared fluorescence imaging
Roderick C Peul
et al
2026
Methods Appl. Fluoresc.
14
027001
View article
, Normalization is essential for accurate intraoperative perfusion assessment using near-infrared fluorescence imaging
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, Normalization is essential for accurate intraoperative perfusion assessment using near-infrared fluorescence imaging
Significance
. Near-infrared fluorescence (NIRF) imaging is increasingly used for perfusion assessment in clinical care and research settings. While subjective interpretation demonstrates clinical benefits, quantitative analysis is crucial for broader adoption and classification of perfusion patterns. However, strict standardization often conflicts with fast-paced clinical workflow, obstructing broad implementation.
Aim and approach
. This study hypothesized that normalization of fluorescence measurements could effectively correct for measurement variability, reducing the need for strict standardization in quantitative NIRF perfusion imaging. To evaluate this, a model capable of consistently simulating fluorescence perfusion patterns was employed. Experiments were conducted in an operating room using four NIRF camera systems under varying measurement conditions.
Results
. Normalization to maximum signal intensity provided consistent perfusion parameter values across differences in camera type, angle, rotation and settings (Δ≤1%). In cases of severe malperfusion where peak-intensity was not reached within the measurement period, normalization did not adequately correct for measurement variability (Δ increased).
Conclusion
. While standardization remains valuable beyond parameter accuracy, appropriate normalization substantially reduces dependence on strict measurement protocols. These findings support broader clinical adoption of quantitative NIRF imaging, extending its utility beyond specialized tertiary centers and facilitating widespread integration into routine surgical care.
The following article is
Open access
Room temperature luminescence of a triangulenium dye ADOTA in PVA films
Zygmunt Gryczynski
et al
2026
Methods Appl. Fluoresc.
14
025003
View article
, Room temperature luminescence of a triangulenium dye ADOTA in PVA films
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, Room temperature luminescence of a triangulenium dye ADOTA in PVA films
The luminescence properties of the cationic triangulenium dye ADOTA embedded in poly(vinyl alcohol) (PVA) were investigated over a broad range of temperatures. We observed extremely efficient delayed fluorescence (DF) with a lifetime of approximately 160 ms. The spectral characteristics of the DF closely match those of the prompt fluorescence. The temperature-dependent emission intensity of ADOTA’s delayed fluorescence reaches a maximum at about 30 °C. This observation reveals for the first time presence of a triplet state T
in triangulenium dyes. Significant intensity and high anisotropy of ADOTA’s DF in time gated detection format offers access to molecular dynamics from micro to millisecond range, in both spectroscopic and microscopic investigations.
The following article is
Open access
Spectral properties of quinine sulfate in PVA films for front-face format emission measurements
Agnieszka Jablonska
et al
2026
Methods Appl. Fluoresc.
14
023001
View article
, Spectral properties of quinine sulfate in PVA films for front-face format emission measurements
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, Spectral properties of quinine sulfate in PVA films for front-face format emission measurements
The luminescence properties of quinine sulfate (QS) embedded in poly(vinyl alcohol) (PVA) film have been studied in detail. Fluorescence excitation and emission spectra were measured using a front-face configuration, and the corresponding intensities were tabulated. A fluorescence quantum yield of 0.51 was determined for QS-doped PVA, using QS dissolved in 1N H
SO
. Both excitation and emission anisotropy measurements indicated effective immobilization of QS molecules within the polymer matrix. The fluorescence lifetime of QS-doped PVA was measured using a picosecond-pulsed 375 nm laser diode and analyzed with both exponential and Lorentzian distribution models. The broad lifetime distribution suggests a complex interaction between QS molecules and the polymer matrix. The QS-doped PVA films are well suited for measurements involving solid materials and front-face geometries.
The following article is
Open access
Four-membered N-heterocyclic carbenes in carbene metal amide emitters: a quantum chemical view
Jasper Guhl
et al
2026
Methods Appl. Fluoresc.
14
025001
View article
, Four-membered N-heterocyclic carbenes in carbene metal amide emitters: a quantum chemical view
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, Four-membered N-heterocyclic carbenes in carbene metal amide emitters: a quantum chemical view
Using computational chemistry, we have scanned a set of four-membered N-heterocyclic carbenes with bulky substituents for their ability to form carbene metal amides (CMAs) with excellent thermally activated delayed fluorescence (TADF) properties. In comparison to the properties of their well-known five- and six-membered analogs, the transition dipole moments of the first excited singlet states of the corresponding Cu
(I)
carbazolide (Cz) complexes increase. For CMAs of the most promising four-membered carbene, a lactam-based carbene (4LAC), detailed investigations of the TADF properties have been performed using advanced quantum chemical methods. Due to the small energy separation between its singlet and triplet ligand-to-ligand charge-transfer (LLCT) states, 4LAC–Ag
(I)
–Cz exhibits the best ratio between reverse intersystem crossing (rISC) and intersystem crossing in the coinage metal triad for a coplanar orientation of the ligands. The TADF properties of the corresponding Cu
(I)
and Au
(I)
complexes benefit from twisted ligand–ligand alignments, achieved by using tetrafluorocarbazolide (4FCz) as donor ligand. The moderate reduction of the fluorescence rate constant upon twisting by about 45–50° is overcompensated by a decrease of the singlet–triplet energy gap, thus improving the TADF performance. Overall, with fluorescence rate constants of the order of 10
−1
and rISC rate constants between 10
and 10
10
−1
, TADF should have competitive advantage over common triplet deactivation processes such as triplet–triplet annihilation. Like in other CMAs, full excited-state geometry relaxation in liquid solution is detrimental for the emission properties. In the solid state, where the formation of a perpendicular ligand–ligand alignment is sterically hindered by the environment, 4LAC–M–Cz and 4LAC–M–4FCz are predicted to be efficient TADF compounds with red to orange emission.
The following article is
Open access
Uranium speciation by time-resolved laser-induced fluorescence spectroscopy
Alena Zavadilová
et al
2026
Methods Appl. Fluoresc.
14
015001
View article
, Uranium speciation by time-resolved laser-induced fluorescence spectroscopy
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, Uranium speciation by time-resolved laser-induced fluorescence spectroscopy
Uranium(VI) speciation in aqueous carbonate solutions was systematically investigated using time-resolved laser-induced fluorescence spectroscopy (TRLFS) across a broad pH range (4.3–13.0) at room temperature. Distinct uranyl complexes were identified based on their luminescence lifetimes and emission spectra, and their formation was correlated with the theoretical speciation models. Particular emphasis was placed on alkaline conditions, where uranium speciation is less understood due to weak luminescence signals. This study revealed the presence of multiple hydroxo and carbonato complexes, including non-luminescent species at high pH. These findings provide new insights into uranium(VI) behaviour in cementitious environments relevant to deep geological repositories. Moreover the dynamics of complex formation were investigated, and the quantity of precipitates were quantified using the methods based on the luminescent properties of uranium and the presence of a complexing agent. The luminescence intensity was shown to be independent of pH and linearly correlated with uranium concentration, confirming TRLFS as a robust tool for uranium quantification in variable geochemical settings. This work contributes to a more accurate understanding of uranium mobility and stability in nuclear waste management scenarios and in locations contaminated with elevated uranium levels as a resulting of ore extraction or processing.
More Open Access articles
Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection
Alexander P Demchenko 2020
Methods Appl. Fluoresc.
022001
View article
, Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection
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, Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection
Photochemical stability is one of the most important parameters that determine the usefulness of organic dyes in different applications. This Review addresses key factors that determine the dye photostability. It is shown that photodegradation can follow different oxygen-dependent and oxygen-independent mechanisms and may involve both
and higher-energy
excited states. Their involvement and contribution depends on dye structure, medium conditions, irradiation power. Fluorescein, rhodamine, BODIPY and cyanine dyes, as well as conjugated polymers are discussed as selected examples illustrating photobleaching mechanisms. The strategies for modulating and improving the photostability are overviewed. They include the improvement of fluorophore design, particularly by attaching protective and anti-fading groups, creating proper medium conditions in liquid, solid and nanoscale environments. The special conditions for biological labeling, sensing and imaging are outlined.
The following article is
Open access
Photophysics of thermally activated delayed fluorescence molecules
Fernando B Dias
et al
2017
Methods Appl. Fluoresc.
012001
View article
, Photophysics of thermally activated delayed fluorescence molecules
PDF
, Photophysics of thermally activated delayed fluorescence molecules
Thermally activated delayed fluorescence (TADF) has recently emerged as one of the most attractive methods for harvesting triplet states in metal-free organic materials for application in organic light emitting diodes (OLEDs). A large number of TADF molecules have been reported in the literature with the purpose of enhancing the efficiency of OLEDs by converting non-emissive triplet states into emissive singlet states. TADF emitters are able to harvest both singlets and triplet states through fluorescence (prompt and delayed), the latter due to the thermally activated reverse intersystem crossing mechanism that allows up-conversion of low energy triplet states to the emissive singlet level. This allows otherwise pure fluorescent OLEDs to overcome their intrinsic limit of 25% internal quantum efficiency (IQE), which is imposed by the 1:3 singlet–triplet ratio arising from the recombination of charges (electrons and holes). TADF based OLEDS with IQEs close to 100% are now routinely fabricated in the green spectral region. There is also significant progress for blue emitters. However, red emitters still show relatively low efficiencies. Despite the significant progress that has been made in recent years, still significant challenges persist to achieve full understanding of the TADF mechanism and improve the stability of these materials. These questions need to be solved in order to fully implement TADF in OLEDs and expand their application to other areas. To date, TADF has been exploited mainly in the field of OLEDs, but applications in other areas, such as sensing and fluorescence microscopies, are envisaged. In this review, the photophysics of TADF molecules is discussed, summarising current methods to characterise these materials and the current understanding of the TADF mechanism in various molecular systems.
Fluorescent J-aggregates of cyanine dyes: basic research and applications review
Julia L Bricks
et al
2018
Methods Appl. Fluoresc.
012001
View article
, Fluorescent J-aggregates of cyanine dyes: basic research and applications review
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, Fluorescent J-aggregates of cyanine dyes: basic research and applications review
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.
Sensing temperature via downshifting emissions of lanthanide-doped metal oxides and salts. A review
Miroslav D Dramićanin 2016
Methods Appl. Fluoresc.
042001
View article
, Sensing temperature via downshifting emissions of lanthanide-doped metal oxides and salts. A review
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, Sensing temperature via downshifting emissions of lanthanide-doped metal oxides and salts. A review
Temperature is important because it has an effect on even the tiniest elements of daily life and is involved in a broad spectrum of human activities. That is why it is the most commonly measured physical quantity. Traditional temperature measurements encounter difficulties when used in some emerging technologies and environments, such as nanotechnology and biomedicine. The problem may be alleviated using optical techniques, one of which is luminescence thermometry. This paper reviews the state of luminescence thermometry and presents different temperature read-out schemes with an emphasis on those utilizing the downshifting emission of lanthanide-doped metal oxides and salts. The read-out schemes for temperature include those based on measurements of spectral characteristics of luminescence (band positions and shapes, emission intensity and ratio of emission intensities), and those based on measurements of the temporal behavior of luminescence (lifetimes and rise times). This review (with 140 references) gives the basics of the fundamental principles and theory that underlie the methods presented, and describes the methodology for the estimation of their performance. The major part of the text is devoted to those lanthanide-doped metal oxides and salts that are used as temperature probes, and to the comparison of their performance and characteristics.
Fluorescence polarization assays in high-throughput screening and drug discovery: a review
Matthew D Hall
et al
2016
Methods Appl. Fluoresc.
022001
View article
, Fluorescence polarization assays in high-throughput screening and drug discovery: a review
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, Fluorescence polarization assays in high-throughput screening and drug discovery: a review
The sensitivity of fluorescence polarization (FP) and fluorescence anisotropy (FA) to molecular weight changes has enabled the interrogation of diverse biological mechanisms, ranging from molecular interactions to enzymatic activity. Assays based on FP/FA technology have been widely utilized in high-throughput screening (HTS) and drug discovery due to the homogenous format, robust performance and relative insensitivity to some types of interferences, such as inner filter effects. Advancements in assay design, fluorescent probes, and technology have enabled the application of FP assays to increasingly complex biological processes. Herein we discuss different types of FP/FA assays developed for HTS, with examples to emphasize the diversity of applicable targets. Furthermore, trends in target and fluorophore selection, as well as assay type and format, are examined using annotated HTS assays within the PubChem database. Finally, practical considerations for the successful development and implementation of FP/FA assays for HTS are provided based on experience at our center and examples from the literature, including strategies for flagging interference compounds among a list of hits.
A review on fluorescence spectroscopic analysis of water and wastewater
Muhammad Farooq Saleem Khan
et al
2022
Methods Appl. Fluoresc.
10
012001
View article
, A review on fluorescence spectroscopic analysis of water and wastewater
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, A review on fluorescence spectroscopic analysis of water and wastewater
In recent years, the application of fluorescence spectroscopy has been widely recognized in water environment studies. The sensitiveness, simplicity, and efficiency of fluorescence spectroscopy are proved to be a promising tool for effective monitoring of water and wastewater. The fluorescence excitation-emission matrix (EEMs) and synchronous fluorescence spectra have been widely used analysis techniques of fluorescence measurement. The presence of organic matter in water and wastewater defines the degree and type of pollution in water. The application of fluorescence spectroscopy to characterize dissolved organic matter (DOM) has made the water quality assessment simple and easy. With the recent advances in this technology, components of DOM are identified by employing parallel factor analysis (PARAFAC), a mathematical trilinear data modeling with EEMs. The majority of wastewater studies indicated that the fluorescence peak of EX/EM at 275 nm/340 nm is referred to tryptophan region (Peak T1). However, some researchers identified another fluorescence peak in the region of EX/EM at 225–237 nm/340–381 nm, which described the tryptophan region and labeled it as Peak T2. Generally, peak T is a protein-like component in the water sample, where T1 and T2 signals were derived from the <0.20
m fraction of pollution. Therefore, a more advanced approach, such as an online fluorescence spectrofluorometer, can be used for the online monitoring of water. The results of various waters studied by fluorescence spectroscopy indicate that changes in peak T intensity could be used for real-time wastewater quality assessment and process control of wastewater treatment works. Finally, due to its effective use in water quality assessment, the fluorescence technique is proved to be a surrogate online monitoring tool and early warning equipment.
Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samples
Marcia Levitus 2020
Methods Appl. Fluoresc.
033001
View article
, Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samples
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, Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samples
The measurement of fluorescence spectra and the determination of fluorescence quantum yields in transparent samples are conceptually simple tasks, but these procedures are subject to several pitfalls that can lead to significant errors. Available technical reports and protocols often assume that the reader possesses a solid theoretical background in spectroscopy and has ample experience with fluorescence instrumentation, but this is often not the case given the many applications of fluorescence in diverse fields of science. The goal of this tutorial is to provide a didactic treatment of the topic that will hopefully be accessible to readers without extensive expertise in the field of fluorescence. The article covers the theoretical background needed to understand the origins of the most common artifacts researchers can expect. Possible artifacts are illustrated with examples to help readers avoid them or identify them if present. A step-by-step example of a fluorescence quantum yield determination in solution is provided with detailed experimental information to help readers understand how to design and analyze experiments.
On the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correction
Joseph Kimball
et al
2020
Methods Appl. Fluoresc.
033002
View article
, On the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correction
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, On the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correction
Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.
Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review
Maksim V Sednev
et al
2015
Methods Appl. Fluoresc.
042004
View article
, Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review
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, Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review
The review deals with commercially available organic dyes possessing large Stokes shifts and their applications as fluorescent labels in optical microscopy based on stimulated emission depletion (STED). STED microscopy breaks Abbe’s diffraction barrier and provides optical resolution beyond the diffraction limit. STED microscopy is non-invasive and requires photostable fluorescent markers attached to biomolecules or other objects of interest. Up to now, in most biology-related STED experiments, bright and photoresistant dyes with small Stokes shifts of 20–40 nm were used. The rapid progress in STED microscopy showed that organic fluorophores possessing large Stokes shifts are indispensable in multi-color super-resolution techniques. The ultimate result of the imaging relies on the optimal combination of a dye, the bio-conjugation procedure and the performance of the optical microscope. Modern bioconjugation methods, basics of STED microscopy, as well as structures and spectral properties of the presently available fluorescent markers are reviewed and discussed. In particular, the spectral properties of the commercial dyes are tabulated and correlated with the available depletion wavelengths found in STED microscopes produced by LEICA Microsytems, Abberior Instruments and Picoquant GmbH.
Sensing with photoluminescent semiconductor quantum dots
Margaret Chern
et al
2019
Methods Appl. Fluoresc.
012005
View article
, Sensing with photoluminescent semiconductor quantum dots
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, Sensing with photoluminescent semiconductor quantum dots
Fluorescent sensors benefit from high signal-to-noise and multiple measurement modalities, enabling a multitude of applications and flexibility of design. Semiconductor nanocrystal quantum dots (QDs) are excellent fluorophores for sensors because of their extraordinary optical properties. They have high thermal and photochemical stability compared to organic dyes or fluorescent proteins and are extremely bright due to their large molar cross-sections. In contrast to organic dyes, QD emission profiles are symmetric, with relatively narrow bandwidths. In addition, the size tunability of their emission color, which is a result of quantum confinement, make QDs exceptional emitters with high color purity from the ultra-violet to near infrared wavelength range. The role of QDs in sensors ranges from simple fluorescent tags, as used in immunoassays, to intrinsic sensors that utilize the inherent photophysical response of QDs to fluctuations in temperature, electric field, or ion concentration. In more complex configurations, QDs and biomolecular recognition moieties like antibodies are combined with a third component to modulate the optical signal via energy transfer. QDs can act as donors, acceptors, or both in energy transfer-based sensors using Förster resonance energy transfer (FRET), nanometal surface energy transfer (NSET), or charge or electron transfer. The changes in both spectral response and photoluminescent lifetimes have been successfully harnessed to produce sensitive sensors and multiplexed devices. While technical challenges related to biofunctionalization and the high cost of laboratory-grade fluorimeters have thus far prevented broad implementation of QD-based sensing in clinical or commercial settings, improvements in bioconjugation methods and detection schemes, including using simple consumer devices like cell phone cameras, are lowering the barrier to broad use of more sensitive QD-based devices.
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2013-present
Methods and Applications in Fluorescence
doi: 10.1088/issn.2050-6120
Online ISSN: 2050-6120