Many ecosystems thrive in near-0 °C conditions, and the mechanisms supporting life in these conditions remain understudied due to the challenges in reproducing such environments in laboratory conditions. One such example is polar organisms, that have adapted their entire lifecycle to operate below freezing temperatures through largely unknown cellular adaptations. As rapid polar warming threatens these species, elucidating their survival strategies is increasingly urgent. Fluorescence-based optical microscopy has been central to the understanding of the dynamic processes sustaining life at the cellular level, yet most imaging approaches have been developed and validated for conditions near mammalian physiological temperatures. Imaging at low temperature introduces a distinct physical regime in which molecular motion, membrane organisation, protein conformational dynamics, and fluorophore photophysics are fundamentally altered. As a result, imaging tools, fluorescent probes, and super-resolution methods optimised at 37 °C often fail when applied near 0 °C, or they report biased information. Here, we examine the conceptual, technical, and practical challenges associated with live-cell fluorescence microscopy at cold temperatures. We discuss when and why common imaging modalities and labelling strategies break down, and how probe behaviour becomes tightly coupled to local changes in physicochemical environment. We offer a perspective on new biological questions that become accessible for study with a microscopy platform optimised for imaging in cold conditions. We highlight trade-offs in current temperature-control strategies and identify unmet needs in fluorophore design, instrument engineering, and quantitative standards. By framing cold microscopy as a distinct operational regime rather than an extension of conventional live-cell imaging, this perspective aims to guide the development of robust tools for studying biological systems near-0 °C conditions.
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- The following article is Open accessChallenges and limitations for live cell imaging in extreme cold
Anne-Pia M Marty et al 2026 Methods Appl. Fluoresc. 14 033101
- The following article is Open accessFused deep learning enables 6D single-molecule localization in polarization-resolved microscopy
Emil Gillett et al 2026 Methods Appl. Fluoresc. 14 035006
View article, Fused deep learning enables 6D single-molecule localization in polarization-resolved microscopyPDF, Fused deep learning enables 6D single-molecule localization in polarization-resolved microscopySingle-molecule orientation localization microscopy (SMOLM) is an optical means to measure complex transport in charged and crowded conditions, such as inside cells or polymer materials. SMOLM extracts time- and space-dependent three-dimensional orientation information from dipole emitters. Achieving simultaneous position-orientation resolution with high photon efficiency remains a central challenge in SMOLM instrument design. We developed an optical fluorescence microscope that uses the double-helix point spread function (DHPSF) to localize dipole emitters in six dimensions (6D), delineated by spatial and dynamic orientational parameters. Furthermore, we developed a fused deep learning approach based on existing neural network architectures to localize dipole emitters in 6D. Our microscope enables simultaneous 6D localization of single fluorophores, achieving a median spatial precision of 10 nm and angular precision below 10° across most of orientation space, except for the azimuthal angle at high polar angles where the DHPSF exhibits known optical degeneracies. We demonstrate our approach by localizing single rhodamine B molecules in poly(methyl methacrylate) films. The recovered orientations show
near 90° and small wobble angles. We also demonstrate 6D SMOLM of a spherical supported lipid bilayer, where despite the low signal, out-of-training distribution of the experimental data, we observe clearly ordered orientation of Nile red molecules within the membrane. - The following article is Open accessUnsupervised clustering FLIM-phasor from multifunctionalized nanoparticles in living cancer cells
Dora-Luz Flores et al 2026 Methods Appl. Fluoresc. 14 035004
View article, Unsupervised clustering FLIM-phasor from multifunctionalized nanoparticles in living cancer cellsPDF, Unsupervised clustering FLIM-phasor from multifunctionalized nanoparticles in living cancer cellsIn the present work, we developed a data-driven algorithm for the automated segmentation of fluorescence lifetime imaging microscopy (FLIM) images, enhancing the analysis of multifunctionalized nanoparticles (NPs) within living cancer cells. FLIM, a powerful microscopy technique, generates images that capture the fluorescence lifetime across a sample at the pixel level, revealing critical details about the molecular environment. Traditionally, FLIM image analysis has relied on manual segmentation with the phasor plot approach, a graphical representation of FLIM data in a G and S coordinate system, which is susceptible to user bias and inconsistency. Here, we present an automated and free of user-biased thresholding and segmentation algorithm that streamlines with clustering techniques to automatically identify phasor-clusters in the phasor plot space, reducing user dependency and providing a reproducible strategy under tested conditions image segmentation. We demonstrate its application in the context of FLIM images displaying a map of intensity heterogeneity where functionalized NPs affect the cellular metabolism of HeLa cells, reported by NADH, a bright and a dim fluorescent source, respectively, both of biological relevance. This algorithm provides a transparent and reproducible approach for FLIM image analysis, showing good agreement with expert-defined segmentation under sufficient contrast conditions, while presenting limitations in low-contrast or noisy regimes.
- The following article is Open accessAn introduction to optical super-resolution microscopy for the adventurous biologist
J Vangindertael et al 2018 Methods Appl. Fluoresc. 6 022003
View article, An introduction to optical super-resolution microscopy for the adventurous biologistPDF, An introduction to optical super-resolution microscopy for the adventurous biologistEver 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 accessSubstituent position-dependent photophysics in fluorinated porphycenes
Katsiaryna Duk et al 2026 Methods Appl. Fluoresc. 14 035003
View article, Substituent position-dependent photophysics in fluorinated porphycenesPDF, Substituent position-dependent photophysics in fluorinated porphycenesMeso-fluorinated porphycenes reveal photophysical properties that vary significantly with the number and position of the substituents. 9,10-difluoro-2,7,12,17-tetra-tert-butylporphycene emits weakly, whereas two derivatives bearing the fluorines on the opposite meso positions: 9,19- and 9,20- exhibit much higher fluorescence quantum yields and longer decay times. For weakly emitting porphycenes, fluorescence can be increased by placing the chromophore in a viscous solvent. The same effect is observed for two novel porphycenes in which a methyl group is placed next to a fluorine: 9-fluoro-2,7-di-tert-butyl-10,19-dimethylporphycene and 9,20-difluoro-2,7-tert-butyl-10,19-dimethylporphycene. These results can be explained by calculations that reveal, in the lowest excited singlet state, a highly nonplanar structure, from which rapid depopulation to S0 can occur. The energy required to attain such geometry is of the order of a few kcal/mol. The relative energies calculated for the three difluorosubstituted porphycenes correlate well with the experimentally obtained fluorescence quantum yields and lifetimes. Based on these results, we propose a model that postulates that the nonradiative deactivation channel in the lowest excited singlet state of porphycenes originates from geometry distortion due to the loss of aromaticity.
- The following article is Open accessPhotophysics of thermally activated delayed fluorescence molecules
Fernando B Dias et al 2017 Methods Appl. Fluoresc. 5 012001
View article, Photophysics of thermally activated delayed fluorescence moleculesPDF, Photophysics of thermally activated delayed fluorescence moleculesThermally 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 accessFluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancement
Peter V Demydov et al 2026 Methods Appl. Fluoresc. 14 032001
View article, Fluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancementPDF, Fluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancementConsidering the recent global growth of terrorism and widespread environmental contamination caused by the previous military actions and ongoing wars, it is evident that the importance of innovation in trace detection of explosives has increased significantly. Specifically, it is vital to develop sensitive, fast, cheap, portable and mass-producible sensors, which will overcome the drawbacks of conventional techniques used for detecting the most widespread explosives, especially those based on nitro compounds. Recently, various optical sensors have been proposed for the detection of explosives due to their real-time, highly sensitive response with significant miniaturization potential. Among them, fluorescence-based sensors have emerged as a promising technique, which can be further improved by employing the plasmon-enhanced fluorescence (PEF) phenomenon for achieving lower detection limits, thus providing the ability to detect different analytes at ultra-low concentrations in a portable format. This paper provides a comprehensive overview of the current state of fluorescence-based trace detection of explosives, highlighting key mechanisms, recent developments and innovations. The prospects of PEF for developing an ultrasensitive portable technique via enhancement provided by plasmonic nanostructures are also discussed, which is important for pursuing innovations in environmental remediation and humanitarian demining.
- The following article is Open accessMicroscale 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 parametersPDF, Microscale thermophoresis as a powerful growing analytical technique for the investigation of biomolecular interaction and the determination of binding parametersThe 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 accessFluorescence Guided Surgery
Hazel L Stewart and David J S Birch 2021 Methods Appl. Fluoresc. 9 042002
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 accessAdvances in ultraviolet microscopy
M McFarlane and G McConnell 2025 Methods Appl. Fluoresc. 13 042001
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.
- A ratiometric fluorescent probe for HSO3− and its application in environmental and food analysis
Chunling Zhou et al 2026 Methods Appl. Fluoresc. 14 045002
View article, A ratiometric fluorescent probe for HSO3− and its application in environmental and food analysisPDF, A ratiometric fluorescent probe for HSO3− and its application in environmental and food analysisBisulfite (HSO3−) is widely used as a preservative and antioxidant in the food industry and chemical manufacturing, yet its excessive residues pose risks to food quality, environmental safety, and human health. To achieve rapid, selective, and quantitative detection of HSO3−, a ratiometric fluorescent probe based on a benzothiazole–carbazole unit and a benzoindole ion was developed. Upon reaction with HSO3−, the probe exhibits a distinct dual-emission response, fluorescence intensity decreases at 612 nm and increases at 467 nm, enabling robust ratiometric sensing (F467/F612). Owing to its pronounced fluorescence modulation (emission signal change > 145 nm) and high sensitivity (limit of detection = 0.62 μM), the probe demonstrates reliable performance in aqueous samples and has been successfully integrated into portable test strips and swabs. Furthermore, when coupled with a smartphone-based colorimetric application software, the probe allows convenient and real-time quantification of both gaseous SO2 and aqueous HSO3− across environmental and food-related matrices. This work thus delivers a practical, instrument-free platform for sulfite monitoring in complex real-world settings.
- Splitting of luminescence bands of doped quantum dot ensembles in composite with polymers on the silver islands films obtained by chemical deposition method
Dmitriy O Sagdeev and Yuriy G Galyametdinov 2026 Methods Appl. Fluoresc. 14 045001
View article, Splitting of luminescence bands of doped quantum dot ensembles in composite with polymers on the silver islands films obtained by chemical deposition methodPDF, Splitting of luminescence bands of doped quantum dot ensembles in composite with polymers on the silver islands films obtained by chemical deposition methodThe study demonstrates significant splitting of the impurity luminescence peak of cadmium sulfide quantum dots (QDs) doped with copper and manganese ions in polymer composites on substrates with silver nanoparticles obtained by chemical deposition. The influence of polymer concentration and QD type on the splitting is studied. Our results demonstrate that the probability of splitting occurrence and the spectral separation between the peaks are governed not by the optical density of the film or the size of the silver islands, but by the inter-island distance, highlighting the critical role of spatial arrangement in the collective optical response of the system. It is hypothesized that the observed effect can be caused by coupling between the luminescent nanoparticles, which have long luminescence lifetimes, and the silver nanoislands, which act as nanoresonators.
- The following article is Open accessFused deep learning enables 6D single-molecule localization in polarization-resolved microscopy
Emil Gillett et al 2026 Methods Appl. Fluoresc. 14 035006
View article, Fused deep learning enables 6D single-molecule localization in polarization-resolved microscopyPDF, Fused deep learning enables 6D single-molecule localization in polarization-resolved microscopySingle-molecule orientation localization microscopy (SMOLM) is an optical means to measure complex transport in charged and crowded conditions, such as inside cells or polymer materials. SMOLM extracts time- and space-dependent three-dimensional orientation information from dipole emitters. Achieving simultaneous position-orientation resolution with high photon efficiency remains a central challenge in SMOLM instrument design. We developed an optical fluorescence microscope that uses the double-helix point spread function (DHPSF) to localize dipole emitters in six dimensions (6D), delineated by spatial and dynamic orientational parameters. Furthermore, we developed a fused deep learning approach based on existing neural network architectures to localize dipole emitters in 6D. Our microscope enables simultaneous 6D localization of single fluorophores, achieving a median spatial precision of 10 nm and angular precision below 10° across most of orientation space, except for the azimuthal angle at high polar angles where the DHPSF exhibits known optical degeneracies. We demonstrate our approach by localizing single rhodamine B molecules in poly(methyl methacrylate) films. The recovered orientations show
near 90° and small wobble angles. We also demonstrate 6D SMOLM of a spherical supported lipid bilayer, where despite the low signal, out-of-training distribution of the experimental data, we observe clearly ordered orientation of Nile red molecules within the membrane. - Effect of particle size and halide composition on structural and optical properties of CsPbX3 nanocrystals
Charu Dubey et al 2026 Methods Appl. Fluoresc. 14 035005
View article, Effect of particle size and halide composition on structural and optical properties of CsPbX3 nanocrystalsPDF, Effect of particle size and halide composition on structural and optical properties of CsPbX3 nanocrystalsCesium lead halide perovskite nanocrystals (NCs) are emerging as a promising material for next-generation optoelectronic systems. This work investigates how variations in particle size and halide composition affect the structural and optical properties of CsPbX3 NCs. CsPbBr3 NCs of different sizes were prepared using a hot-injection procedure, with the ability to regulate the quenching time, and it was found that larger NCs have a smaller band gap. The crystal structure was found to be cubic. The influence of the halide composition was analyzed by synthesizing CsPbCl3, CsPbBr3, and CsPbI3 under the same conditions. CsPbCl3 showed the broadest bandgap and shortest lifetime, CsPbI3 the closest bandgap and longest lifetime, and CsPbBr3 showed a good compromise between bandgap, luminescence intensity, and structural stability. Optical tuning was also confirmed with respect to size and composition, with reference to UV–visible absorption and photoluminescence tests; blue, green, and red emissions were observed in the case of CsPbCl3, CsPbBr3, and CsPbI3, respectively, with UV illumination. These findings demonstrate the synergistic nature of particle size and halide composition in designing the structural and optical characteristics of CsPbX3 NCs to offer principles of perovskite-based devices.
- The following article is Open accessChallenges and limitations for live cell imaging in extreme cold
Anne-Pia M Marty et al 2026 Methods Appl. Fluoresc. 14 033101
View article, Challenges and limitations for live cell imaging in extreme coldPDF, Challenges and limitations for live cell imaging in extreme coldMany ecosystems thrive in near-0 °C conditions, and the mechanisms supporting life in these conditions remain understudied due to the challenges in reproducing such environments in laboratory conditions. One such example is polar organisms, that have adapted their entire lifecycle to operate below freezing temperatures through largely unknown cellular adaptations. As rapid polar warming threatens these species, elucidating their survival strategies is increasingly urgent. Fluorescence-based optical microscopy has been central to the understanding of the dynamic processes sustaining life at the cellular level, yet most imaging approaches have been developed and validated for conditions near mammalian physiological temperatures. Imaging at low temperature introduces a distinct physical regime in which molecular motion, membrane organisation, protein conformational dynamics, and fluorophore photophysics are fundamentally altered. As a result, imaging tools, fluorescent probes, and super-resolution methods optimised at 37 °C often fail when applied near 0 °C, or they report biased information. Here, we examine the conceptual, technical, and practical challenges associated with live-cell fluorescence microscopy at cold temperatures. We discuss when and why common imaging modalities and labelling strategies break down, and how probe behaviour becomes tightly coupled to local changes in physicochemical environment. We offer a perspective on new biological questions that become accessible for study with a microscopy platform optimised for imaging in cold conditions. We highlight trade-offs in current temperature-control strategies and identify unmet needs in fluorophore design, instrument engineering, and quantitative standards. By framing cold microscopy as a distinct operational regime rather than an extension of conventional live-cell imaging, this perspective aims to guide the development of robust tools for studying biological systems near-0 °C conditions.
- The following article is Open accessFluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancement
Peter V Demydov et al 2026 Methods Appl. Fluoresc. 14 032001
View article, Fluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancementPDF, Fluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancementConsidering the recent global growth of terrorism and widespread environmental contamination caused by the previous military actions and ongoing wars, it is evident that the importance of innovation in trace detection of explosives has increased significantly. Specifically, it is vital to develop sensitive, fast, cheap, portable and mass-producible sensors, which will overcome the drawbacks of conventional techniques used for detecting the most widespread explosives, especially those based on nitro compounds. Recently, various optical sensors have been proposed for the detection of explosives due to their real-time, highly sensitive response with significant miniaturization potential. Among them, fluorescence-based sensors have emerged as a promising technique, which can be further improved by employing the plasmon-enhanced fluorescence (PEF) phenomenon for achieving lower detection limits, thus providing the ability to detect different analytes at ultra-low concentrations in a portable format. This paper provides a comprehensive overview of the current state of fluorescence-based trace detection of explosives, highlighting key mechanisms, recent developments and innovations. The prospects of PEF for developing an ultrasensitive portable technique via enhancement provided by plasmonic nanostructures are also discussed, which is important for pursuing innovations in environmental remediation and humanitarian demining.
- 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 characteristicsPDF, A review on application of laser induced fluorescence spectroscopy in exploring bioaerosol characteristicsAtmospheric 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 accessSpectral 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 measurementsPDF, Spectral properties of quinine sulfate in PVA films for front-face format emission measurementsThe 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 H2SO4. 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 accessAdvances in ultraviolet microscopy
M McFarlane and G McConnell 2025 Methods Appl. Fluoresc. 13 042001
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 accessFiber-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 conceptsPDF, Fiber-optics based fluorescence detection. Part I: Basic conceptsContinuous 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 accessEvaluating the Far-red Emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) Chemogenetic Labeling System for Live-Cell Super-Resolution Microscopy
Kozma et al
View accepted manuscript, Evaluating the Far-red Emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) Chemogenetic Labeling System for Live-Cell Super-Resolution MicroscopyPDF, Evaluating the Far-red Emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) Chemogenetic Labeling System for Live-Cell Super-Resolution MicroscopyFluorogen-activating proteins (FAPs) provide renewable fluorescence signals through continuous fluorogen exchange, resulting in imaging that is virtually resistant to photobleaching, an especially appealing feature for deterministic super-resolution microscopy techniques. Furthermore, careful adjustment of the fluorogen concentration may lead to reversible, exchange-driven stochastic blinking, allowing the use of FAPs in stochastic superresolution microscopy methods. The green-light-excitable, far-red-emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) is particularly well suited for biological imaging due to its favorable spectral properties and live-cell compatibility, however, its performance across different super-resolution microscopy (SRM) modalities has not been evaluated. Here, we assess frFAST for single-molecule localization microscopy (SMLM), fluctuation-based computational approaches (super-resolution optical fluctuation imaging -SOFI and extended super-resolution radial fluctuations -eSRRF), and stimulated emission depletion (STED) microscopy. By tuning fluorogen concentration, we induced reversible stochastic blinking in fixed and live cells, enabling SMLM imaging of cytoskeletal structures and filopodia in mammalian and neuroblastoma cells, without the need for harsh reducing agents. Additionally, we successfully labeled and imaged mitochondrial outer membrane, microtubules, cytoskeleton and histone proteins in live cell STED microscopy. While the renewable nature of the frFAST:HPAR-3OM interaction supports extended live-cell imaging for up to 40 minutes with limited photobleaching, we found that its performance is strongly modality dependent. In particular, intrinsic photophysical properties limit its suitability for classical SMLM, whereas fluctuation-based methods and live-cell STED microscopy are more compatible with frFAST.
- A novel rhodamine-based dual-mode fluorogenic probe for sensitive determination of Hg²⁺
Liu et al
View accepted manuscript, A novel rhodamine-based dual-mode fluorogenic probe for sensitive determination of Hg²⁺PDF, A novel rhodamine-based dual-mode fluorogenic probe for sensitive determination of Hg²⁺Mercury ions (Hg2+) are highly toxic and persistent heavy metal pollutants that pose serious threats to environmental systems and human health. Therefore, developing sensitive and selective detection strategies for Hg2+ is of great significance. In this study, a rhodamine-based dual-mode fluorescent probe (RB-HG) was developed utilizing dimethylthiocarbamate groups as recognition sites. Upon undergoing specific interaction with Hg²⁺, leading to the dissociation of the C-O bond and subsequently triggering an opening-ring reaction to form a ketone structure (RB-C=O), thereby activating the intramolecular charge transfer (ICT) process and generating both fluorescence and colorimetric signals, thereby activating both colorimetric and fluorescence dual - channel signals. In vitro experiments demonstrate that RB-HG (5.0 μM) exhibits high selectivity and sensitivity toward Hg²⁺. The limits of detection for the fluorescence and colorimetric detection modes are 1.16 μM and 3.22 μM, respectively. RB-HG was successfully applied to detect Hg2+ in food samples with excellent recovery rates and to image exogenous Hg2+ in living cells, highlighting its potential for food monitoring and bioimaging applications.
- The following article is Open accessExperimental and simulated FRAP for the quantitative determination of protein diffusion in helical cells
Sakib et al
View accepted manuscript, Experimental and simulated FRAP for the quantitative determination of protein diffusion in helical cellsPDF, Experimental and simulated FRAP for the quantitative determination of protein diffusion in helical cellsFluorescence recovery after photobleaching (FRAP) is widely used to characterize diffusion in cells, but quantitative interpretation of the data in small prokaryotes requires explicitly accounting for cell geometry. While this has been successfully achieved for spherical and rod-shaped bacteria, analytical approaches developed in these cases are not directly applicable to cells with more complex morphologies. Here, we explore the application of FRAP to helical bacteria using simulations. We show that half-compartment FRAP experiments, where one-half of the cell is photobleached, provide a robust means of characterizing fast protein diffusion. To help with the practical implementation of this technique, we established the relationship between the diffusion coefficient and characteristic fluorescence recovery time as a function of cell length and helical parameters, and for two different ways of estimating the recovery time. As a first application, we report measurements of the diffusion coefficient of the fluorescent protein, mNeonGreen, in the helical bacterium Paramagnetospirillum magneticum AMB-1. We find it to be D = 4.9 ± 2.2 μm2.s-1 in isosmotic conditions, not significantly different from the value measured in Escherichia coli. Although developed for helical bacteria, including spirilla, spirochetes, and vibrios, our framework can readily be extended to cells or compartments with other geometries.
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- The following article is Open accessEvaluating the Far-red Emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) Chemogenetic Labeling System for Live-Cell Super-Resolution Microscopy
Eszter Kozma et al 2026 Methods Appl. Fluoresc.
View article, Evaluating the Far-red Emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) Chemogenetic Labeling System for Live-Cell Super-Resolution MicroscopyPDF, Evaluating the Far-red Emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) Chemogenetic Labeling System for Live-Cell Super-Resolution MicroscopyFluorogen-activating proteins (FAPs) provide renewable fluorescence signals through continuous fluorogen exchange, resulting in imaging that is virtually resistant to photobleaching, an especially appealing feature for deterministic super-resolution microscopy techniques. Furthermore, careful adjustment of the fluorogen concentration may lead to reversible, exchange-driven stochastic blinking, allowing the use of FAPs in stochastic superresolution microscopy methods. The green-light-excitable, far-red-emitting Fluorescence-Activating and Absorption-Shifting Tag (frFAST) is particularly well suited for biological imaging due to its favorable spectral properties and live-cell compatibility, however, its performance across different super-resolution microscopy (SRM) modalities has not been evaluated. Here, we assess frFAST for single-molecule localization microscopy (SMLM), fluctuation-based computational approaches (super-resolution optical fluctuation imaging -SOFI and extended super-resolution radial fluctuations -eSRRF), and stimulated emission depletion (STED) microscopy. By tuning fluorogen concentration, we induced reversible stochastic blinking in fixed and live cells, enabling SMLM imaging of cytoskeletal structures and filopodia in mammalian and neuroblastoma cells, without the need for harsh reducing agents. Additionally, we successfully labeled and imaged mitochondrial outer membrane, microtubules, cytoskeleton and histone proteins in live cell STED microscopy. While the renewable nature of the frFAST:HPAR-3OM interaction supports extended live-cell imaging for up to 40 minutes with limited photobleaching, we found that its performance is strongly modality dependent. In particular, intrinsic photophysical properties limit its suitability for classical SMLM, whereas fluctuation-based methods and live-cell STED microscopy are more compatible with frFAST.
- The following article is Open accessExperimental and simulated FRAP for the quantitative determination of protein diffusion in helical cells
Shariful Sakib and Cecile Fradin 2026 Methods Appl. Fluoresc.
View article, Experimental and simulated FRAP for the quantitative determination of protein diffusion in helical cellsPDF, Experimental and simulated FRAP for the quantitative determination of protein diffusion in helical cellsFluorescence recovery after photobleaching (FRAP) is widely used to characterize diffusion in cells, but quantitative interpretation of the data in small prokaryotes requires explicitly accounting for cell geometry. While this has been successfully achieved for spherical and rod-shaped bacteria, analytical approaches developed in these cases are not directly applicable to cells with more complex morphologies. Here, we explore the application of FRAP to helical bacteria using simulations. We show that half-compartment FRAP experiments, where one-half of the cell is photobleached, provide a robust means of characterizing fast protein diffusion. To help with the practical implementation of this technique, we established the relationship between the diffusion coefficient and characteristic fluorescence recovery time as a function of cell length and helical parameters, and for two different ways of estimating the recovery time. As a first application, we report measurements of the diffusion coefficient of the fluorescent protein, mNeonGreen, in the helical bacterium Paramagnetospirillum magneticum AMB-1. We find it to be D = 4.9 ± 2.2 μm2.s-1 in isosmotic conditions, not significantly different from the value measured in Escherichia coli. Although developed for helical bacteria, including spirilla, spirochetes, and vibrios, our framework can readily be extended to cells or compartments with other geometries.
- The following article is Open accessFused deep learning enables 6D single-molecule localization in polarization-resolved microscopy
Emil Gillett et al 2026 Methods Appl. Fluoresc. 14 035006
View article, Fused deep learning enables 6D single-molecule localization in polarization-resolved microscopyPDF, Fused deep learning enables 6D single-molecule localization in polarization-resolved microscopySingle-molecule orientation localization microscopy (SMOLM) is an optical means to measure complex transport in charged and crowded conditions, such as inside cells or polymer materials. SMOLM extracts time- and space-dependent three-dimensional orientation information from dipole emitters. Achieving simultaneous position-orientation resolution with high photon efficiency remains a central challenge in SMOLM instrument design. We developed an optical fluorescence microscope that uses the double-helix point spread function (DHPSF) to localize dipole emitters in six dimensions (6D), delineated by spatial and dynamic orientational parameters. Furthermore, we developed a fused deep learning approach based on existing neural network architectures to localize dipole emitters in 6D. Our microscope enables simultaneous 6D localization of single fluorophores, achieving a median spatial precision of 10 nm and angular precision below 10° across most of orientation space, except for the azimuthal angle at high polar angles where the DHPSF exhibits known optical degeneracies. We demonstrate our approach by localizing single rhodamine B molecules in poly(methyl methacrylate) films. The recovered orientations show
near 90° and small wobble angles. We also demonstrate 6D SMOLM of a spherical supported lipid bilayer, where despite the low signal, out-of-training distribution of the experimental data, we observe clearly ordered orientation of Nile red molecules within the membrane. - The following article is Open accessChallenges and limitations for live cell imaging in extreme cold
Anne-Pia M Marty et al 2026 Methods Appl. Fluoresc. 14 033101
View article, Challenges and limitations for live cell imaging in extreme coldPDF, Challenges and limitations for live cell imaging in extreme coldMany ecosystems thrive in near-0 °C conditions, and the mechanisms supporting life in these conditions remain understudied due to the challenges in reproducing such environments in laboratory conditions. One such example is polar organisms, that have adapted their entire lifecycle to operate below freezing temperatures through largely unknown cellular adaptations. As rapid polar warming threatens these species, elucidating their survival strategies is increasingly urgent. Fluorescence-based optical microscopy has been central to the understanding of the dynamic processes sustaining life at the cellular level, yet most imaging approaches have been developed and validated for conditions near mammalian physiological temperatures. Imaging at low temperature introduces a distinct physical regime in which molecular motion, membrane organisation, protein conformational dynamics, and fluorophore photophysics are fundamentally altered. As a result, imaging tools, fluorescent probes, and super-resolution methods optimised at 37 °C often fail when applied near 0 °C, or they report biased information. Here, we examine the conceptual, technical, and practical challenges associated with live-cell fluorescence microscopy at cold temperatures. We discuss when and why common imaging modalities and labelling strategies break down, and how probe behaviour becomes tightly coupled to local changes in physicochemical environment. We offer a perspective on new biological questions that become accessible for study with a microscopy platform optimised for imaging in cold conditions. We highlight trade-offs in current temperature-control strategies and identify unmet needs in fluorophore design, instrument engineering, and quantitative standards. By framing cold microscopy as a distinct operational regime rather than an extension of conventional live-cell imaging, this perspective aims to guide the development of robust tools for studying biological systems near-0 °C conditions.
- The following article is Open accessUnsupervised clustering FLIM-phasor from multifunctionalized nanoparticles in living cancer cells
Dora-Luz Flores et al 2026 Methods Appl. Fluoresc. 14 035004
View article, Unsupervised clustering FLIM-phasor from multifunctionalized nanoparticles in living cancer cellsPDF, Unsupervised clustering FLIM-phasor from multifunctionalized nanoparticles in living cancer cellsIn the present work, we developed a data-driven algorithm for the automated segmentation of fluorescence lifetime imaging microscopy (FLIM) images, enhancing the analysis of multifunctionalized nanoparticles (NPs) within living cancer cells. FLIM, a powerful microscopy technique, generates images that capture the fluorescence lifetime across a sample at the pixel level, revealing critical details about the molecular environment. Traditionally, FLIM image analysis has relied on manual segmentation with the phasor plot approach, a graphical representation of FLIM data in a G and S coordinate system, which is susceptible to user bias and inconsistency. Here, we present an automated and free of user-biased thresholding and segmentation algorithm that streamlines with clustering techniques to automatically identify phasor-clusters in the phasor plot space, reducing user dependency and providing a reproducible strategy under tested conditions image segmentation. We demonstrate its application in the context of FLIM images displaying a map of intensity heterogeneity where functionalized NPs affect the cellular metabolism of HeLa cells, reported by NADH, a bright and a dim fluorescent source, respectively, both of biological relevance. This algorithm provides a transparent and reproducible approach for FLIM image analysis, showing good agreement with expert-defined segmentation under sufficient contrast conditions, while presenting limitations in low-contrast or noisy regimes.
- The following article is Open accessSubstituent position-dependent photophysics in fluorinated porphycenes
Katsiaryna Duk et al 2026 Methods Appl. Fluoresc. 14 035003
View article, Substituent position-dependent photophysics in fluorinated porphycenesPDF, Substituent position-dependent photophysics in fluorinated porphycenesMeso-fluorinated porphycenes reveal photophysical properties that vary significantly with the number and position of the substituents. 9,10-difluoro-2,7,12,17-tetra-tert-butylporphycene emits weakly, whereas two derivatives bearing the fluorines on the opposite meso positions: 9,19- and 9,20- exhibit much higher fluorescence quantum yields and longer decay times. For weakly emitting porphycenes, fluorescence can be increased by placing the chromophore in a viscous solvent. The same effect is observed for two novel porphycenes in which a methyl group is placed next to a fluorine: 9-fluoro-2,7-di-tert-butyl-10,19-dimethylporphycene and 9,20-difluoro-2,7-tert-butyl-10,19-dimethylporphycene. These results can be explained by calculations that reveal, in the lowest excited singlet state, a highly nonplanar structure, from which rapid depopulation to S0 can occur. The energy required to attain such geometry is of the order of a few kcal/mol. The relative energies calculated for the three difluorosubstituted porphycenes correlate well with the experimentally obtained fluorescence quantum yields and lifetimes. Based on these results, we propose a model that postulates that the nonradiative deactivation channel in the lowest excited singlet state of porphycenes originates from geometry distortion due to the loss of aromaticity.
- The following article is Open accessFluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancement
Peter V Demydov et al 2026 Methods Appl. Fluoresc. 14 032001
View article, Fluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancementPDF, Fluorescence-based trace detection of explosives: current state and perspective for ultrasensitive portable technique via plasmonic enhancementConsidering the recent global growth of terrorism and widespread environmental contamination caused by the previous military actions and ongoing wars, it is evident that the importance of innovation in trace detection of explosives has increased significantly. Specifically, it is vital to develop sensitive, fast, cheap, portable and mass-producible sensors, which will overcome the drawbacks of conventional techniques used for detecting the most widespread explosives, especially those based on nitro compounds. Recently, various optical sensors have been proposed for the detection of explosives due to their real-time, highly sensitive response with significant miniaturization potential. Among them, fluorescence-based sensors have emerged as a promising technique, which can be further improved by employing the plasmon-enhanced fluorescence (PEF) phenomenon for achieving lower detection limits, thus providing the ability to detect different analytes at ultra-low concentrations in a portable format. This paper provides a comprehensive overview of the current state of fluorescence-based trace detection of explosives, highlighting key mechanisms, recent developments and innovations. The prospects of PEF for developing an ultrasensitive portable technique via enhancement provided by plasmonic nanostructures are also discussed, which is important for pursuing innovations in environmental remediation and humanitarian demining.
- The following article is Open accessCompensating for photon counting losses in a TCSPC SPAD array enables quantitative time-resolved fluorescence anisotropy imaging
Louis Obeid Mogridge et al 2026 Methods Appl. Fluoresc. 14 035001
View article, Compensating for photon counting losses in a TCSPC SPAD array enables quantitative time-resolved fluorescence anisotropy imagingPDF, Compensating for photon counting losses in a TCSPC SPAD array enables quantitative time-resolved fluorescence anisotropy imagingWe devise and experimentally validate a theoretical model to account for lost photon counts during the exposure time of a time-correlated single photon counting (TCSPC)-based QuantICAM single photon avalanche diode array camera. The work is motivated by the quest for TCSPC-based wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM), implemented by the acquisition of images at orthogonal polarization. For accurate, quantitatively correct TR-FAIM, the two images must be acquired under equivalent conditions and any photons lost during the camera exposures must be precisely quantified. Our model is based on a binomial distribution with a single adjustable parameter. We plot the recorded versus the true photon counts for exposure times of 250 µs and 1000 µs, using photons with random arrival times and from fluorescence decays. Our model describes the experimental data well and the correct number of excitation cycles during the exposure time is extracted from least-squares fits of the binomial model to the experimental data. On the basis of this model, we account for lost photons in TCSPC-based TR-FAIM and show that a compensation for lost photons is essential to obtain quantitatively correct steady-state anisotropy and G-factor histograms in TR-FAIM. We also show that, under the conditions used, the rotational correlation time, initial anisotropy r0 and hindered rotation parameter
histograms are only marginally affected by lost photons. Our work thus paves the way for robust and reliable TCSPC-based TR-FAIM. - The following article is Open accessSelective luminescence enhancement of lanthanide complexes using diffraction gratings
Derek A Russell et al 2026 Methods Appl. Fluoresc. 14 025010
View article, Selective luminescence enhancement of lanthanide complexes using diffraction gratingsPDF, Selective luminescence enhancement of lanthanide complexes using diffraction gratingsLinear and superimposed diffraction gratings have been designed to selectively enhance light emission at specific wavelengths from thin films of lanthanide complexes at multiple luminescent transitions. Optimal grating pitches were determined through numerical modeling and the gratings were fabricated on photoactive azobenzene films, then transferred through nano-imprint lithography, to a bilayer of a europium dye-doped polymethyl methacrylate thin film mounted on an epoxy layer. The optimized gratings were found to increase emission intensity by up to ninefold at the emission wavelengths through angle-dependent out-coupling of waveguiding modes. This strategy was extended to simultaneously increase the emission intensity of multiple bands of the europium dye at the emission wavelengths by using a triple parallel-superimposed grating. This work demonstrates how diffraction gratings on thin films can be readily designed and manufactured for tunable angular control and wavelength-specific enhancement of emission bands from thin films doped with dyes.
- The following article is Open accessSterol 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. 14 025009
View article, Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analogPDF, Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analogErgosterol 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 liquid-ordered 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.
- Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection
Alexander P Demchenko 2020 Methods Appl. Fluoresc. 8 022001
View article, Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protectionPDF, Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protectionPhotochemical 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 1S1–3T1 and higher-energy 1Sn–3Tn 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 accessPhotophysics of thermally activated delayed fluorescence molecules
Fernando B Dias et al 2017 Methods Appl. Fluoresc. 5 012001
View article, Photophysics of thermally activated delayed fluorescence moleculesPDF, Photophysics of thermally activated delayed fluorescence moleculesThermally 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. 6 012001
View article, Fluorescent J-aggregates of cyanine dyes: basic research and applications reviewPDF, Fluorescent J-aggregates of cyanine dyes: basic research and applications reviewJ-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. 4 042001
View article, Sensing temperature via downshifting emissions of lanthanide-doped metal oxides and salts. A reviewPDF, Sensing temperature via downshifting emissions of lanthanide-doped metal oxides and salts. A reviewTemperature 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. 4 022001
View article, Fluorescence polarization assays in high-throughput screening and drug discovery: a reviewPDF, Fluorescence polarization assays in high-throughput screening and drug discovery: a reviewThe 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.
- Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samples
Marcia Levitus 2020 Methods Appl. Fluoresc. 8 033001
View article, Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samplesPDF, Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samplesThe 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.
- 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 wastewaterPDF, A review on fluorescence spectroscopic analysis of water and wastewaterIn 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 μm 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.
- 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. 8 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 correctionPDF, On the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correctionFluorescence 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. 3 042004
View article, Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a reviewPDF, Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a reviewThe 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.
- The following article is Open accessDynamic and static quenching of 2-aminopurine fluorescence by the natural DNA nucleotides in solution
Kyle A Paterson et al 2020 Methods Appl. Fluoresc. 8 025002
View article, Dynamic and static quenching of 2-aminopurine fluorescence by the natural DNA nucleotides in solutionPDF, Dynamic and static quenching of 2-aminopurine fluorescence by the natural DNA nucleotides in solution2-aminopurine (2AP) is a responsive fluorescent base analogue that is used widely as a probe of the local molecular environment in DNA. The ability of 2AP to report changes in local conformation and base-stacking interactions arises from the efficient quenching of its fluorescence by the natural DNA bases. However, the mechanism of this inter-base quenching remains imperfectly understood. Two previous studies of the collisional quenching of 2AP by the natural bases, in different buffer solutions, showed that dynamic quenching efficiency depends on the identity of the natural base, but disagreed on the relative quenching efficiencies of the bases. We report a comprehensive investigation of inter-base quenching of 2AP by the natural nucleoside monophosphates (NMPs), replicating the buffer conditions used in the previous studies. Using time-resolved fluorescence measurements to distinguish between dynamic and static quenching, we find that the dynamic quenching rate constants of the different bases show a consistent trend across both buffers, and this is in line with a charge-transfer mechanism. Time-resolved measurements also provide insight into static quenching, revealing formation of 2AP-NMP ground-state complexes in which 2AP displays a very short fluorescence lifetime, comparable to that seen in oligonucleotides. In these complexes, the dependence of the rate of quenching on the partner base also supports a charge-transfer mechanism.
Journal resources
Journal information
- 2013-present
Methods and Applications in Fluorescence
doi: 10.1088/issn.2050-6120
Online ISSN: 2050-6120