Methods and Applications in Fluorescence

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 resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machine in situ, i.e., the injectisome of the enteropathogen Y. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule level in situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.

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.

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 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.

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.

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.

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.

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.

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.

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analog in DNA and RNA research. While quenching of 2AP by DNA bases has been extensively characterized, the effect of extrinsic quenchers has received far less attention. This study examines the fluorescence quenching mechanisms of 2AP by commonly used buffers in biochemical research. We systematically investigated four Good’s buffers—MES, MOPS, HEPES, and PIPES—along with their parent compounds morpholine and piperazine across a range of pH conditions and concentrations. For morpholine-containing buffers (MES and MOPS), quenching occurs predominantly at pH values at or above their respective pKa values and is negligible at more than two pH units below the pKa. In contrast, piperazine-containing buffers (HEPES and PIPES) exhibit substantial quenching even below their pKa values due to the presence of two basic nitrogen atoms in the piperazine ring, one of which remains unprotonated and reactive across the investigated pH range. Time-resolved fluorescence measurements demonstrate that quenching is primarily dynamic for MES, MOPS, and HEPES, while PIPES shows significant static quenching contributions. Results are consistent with a mechanism involving photoinduced electron transfer from unprotonated tertiary amines to excited-state 2AP. The thermodynamic feasibility of this mechanism is supported by the low oxidation potentials of these tertiary amines compared to primary amine-containing buffers such as TRIS, which does not quench 2AP fluorescence. These results have significant practical implications for fluorescence-based studies using 2AP as a structural or dynamic probe in nucleic acid research. Buffer selection can substantially alter both quantum yields and fluorescence lifetimes of 2AP, potentially leading to misinterpretation of experimental data if these effects are not properly accounted for.

The fluorescent on-chip imaging system differs from a conventional fluorescent microscope in terms of the imaging method because the sample is directly placed on the imaging sensor (i.e., charge-coupled device (CCD)). While this imaging modality presents several advantages, including a wide field of view and rapid scanning speed, it can be difficult to detect certain particles in dense and scattering environments, such as whole blood and tissue. These difficulties lead to a decreased signal-to-noise ratio (SNR) in the captured images, influenced by both the medium’s light-transmitting capability and the excitation techniques used. In this paper, we quantitatively examine the effect of beam shaping techniques on a fluorescent on-chip imaging system from the SNR perspective. An experimental comparison is conducted between a Gaussian beam and plane-wave illumination generated by a novel phase modulation schema using our developed imaging platform. The results indicate that the Gaussian beam produces higher SNR images than plane waves when detecting fluorescent particles in a microchannel. Gaussian beam’s higher energy confinement ability enhances the image quality of on-chip fluorescent imaging systems, particularly involving scattering-like medium limitations.

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.

Fluorescence-based optical techniques are developing rapidly, giving access to high spatiotemporal information on live biological systems with single molecule sensitivity. However, these techniques are typically restricted to expert labs and are not easily accessible to the general user. While the development of customized systems and their wider distribution is difficult, as it requires expert manpower, software developments are easy to distribute. However, in reality only few users outside an expert community are exploring and using these tools. This is due to the usability of the software which often requires expert skills to operate and is neither intuitive nor easy to use. These issues of accessibility and usability limit the spread of state-of-the-art techniques. And while accessibility of custom instrumentation is difficult to solve, the accessibility and usability of software is an easier target. In this perspective, therefore, we concentrate on the software issue and examine the major translational barriers that prevent biologists from adopting the available fluorescence microscopy techniques. We discuss key developments in the field such as open-source tools, standardized file formats and AI-driven analysis platforms, and suggest a roadmap to bring advanced tools to a wider community.

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.

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 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.

In the past, there were limited efforts to use light-emitting diodes (LEDs) for pumping solid-state lasers. However, these attempts were overshadowed by the introduction of laser diodes, which offered more favourable pumping conditions. Nevertheless, recent advancements in high-power LEDs, coupled with the utilization of luminescent concentrators (LC), have paved the way for a novel approach to pump solid-state lasers. The combination of LEDs and LC in this LED-LC system presents several advantages, including enhanced ruggedness, stability, and cost-effectiveness compared to other laser pumping methods. This review explores the various techniques employed to pump solid-state lasers using LED-LC as a pump source, along with improvements made to enhance the brightness of LEDs in this context.

Many medical imaging modalities have benefited from recent advances in Machine Learning (ML), specifically in deep learning, such as neural networks. Computers can be trained to investigate and enhance medical imaging methods without using valuable human resources. In recent years, Fluorescence Lifetime Imaging (FLIm) has received increasing attention from the ML community. FLIm goes beyond conventional spectral imaging, providing additional lifetime information, and could lead to optical histopathology supporting real-time diagnostics. However, most current studies do not use the full potential of machine/deep learning models. As a developing image modality, FLIm data are not easily obtainable, which, coupled with an absence of standardisation, is pushing back the research to develop models which could advance automated diagnosis and help promote FLIm. In this paper, we describe recent developments that improve FLIm image quality, specifically time-domain systems, and we summarise sensing, signal-to-noise analysis and the advances in registration and low-level tracking. We review the two main applications of ML for FLIm: lifetime estimation and image analysis through classification and segmentation. We suggest a course of action to improve the quality of ML studies applied to FLIm. Our final goal is to promote FLIm and attract more ML practitioners to explore the potential of lifetime imaging.

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 10⁷ s⁻¹ and rISC rate constants between 10⁹ and 10¹⁰ s⁻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.

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 10⁷ s⁻¹ and rISC rate constants between 10⁹ and 10¹⁰ s⁻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.

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.

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.

Accurate and efficient autofocusing is essential for the automation of fluorescence microscopy, but background noise and shallow depth of field at high magnifications make autofocusing particularly challenging. Here, we present a fast and accurate autofocus algorithm to address these challenges. It is highly effective for high-magnification imaging, while performing equally well for low-magnification imaging tasks. The method is based on the mountain climbing search algorithm and yields improvements on autofocusing precision of up to 200-fold over current methods, whilst offering competitive speed and greatly extended search ranges. Our approach is broadly applicable: it demonstrated good stability and reproducibility across magnifications ranging from 20X to 100X, excels in both live cell imaging and high-resolution fixed sample imaging, and it is compatible with various microscopy techniques without the need for fiducial markers or hardware modifications on existing microscopes. To maximise its accessibility, we constructed a user-friendly interface compatible with the widely used Micromanager software. It generalises well across various imaging modalities and hardware platforms, making it particularly suitable for use in high-resolution screening of candidate drugs.

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.

This study explores the fluorescence enhancement of quantum dots (QDs) by gold film over nanospheres (AuFoN) plasmonic substrates, focusing on how a polymer matrix and plasmon resonances of the substrate affect the fluorescence properties of QDs. It was observed that polyvinylpyrrolidone (PVP) facilitated the uniform distribution of QDs on the surface of the AuFoN by simple drop-coating, avoiding aggregation during solvent evaporation. Progressive fluorescence redshifts and intensity enhancement were observed when moving from QDs on glass substrates to planar Au, and most pronouncedly, to nanostructured AuFoN substrates. The fluorescence enhancement was further analyzed by varying the diameter of the polystyrene spheres used in AuFoN fabrication, revealing that substrates based on 600–700 nm spheres provided the strongest fluorescence amplification due to stronger localized electromagnetic fields. Time-resolved fluorescence measurements revealed two primary fluorescence lifetime components for QDs on AuFoN: a short component linked to non-radiative plasmonic energy transfer and a long component representing intrinsic QDs emission. By optimizing sphere size, Au nanostructured films can be tailored to control QDs fluorescence lifetimes and intensity, advancing their use in biosensing, photonics, and other fluorescence-based technologies. This work enhances our understanding of how substrate design and matrix effects impact QDs fluorescence, providing a pathway for precisely engineered Surface Enhanced Fluorescence (SEF) platforms suited to various applications in optical sensing and more general photonics.

We studied absorption and fluorescence as well as room temperature phosphorescence (RTP) of 4-methylumbelliferone (4MU) in poly (vinyl alcohol) (PVA) films. We focused our study on the long-wavelength basic form of 4MU with absorption centered at 375 nm. The strong fluorescence with a quantum yield of above 70% appears at ∼430 nm. The fluorescence anisotropy of 4MU-doped PVA film is high, reaching a value of about 0.3. The emission with gated detection shows a broad phosphorescence spectrum with a peak at ∼510 nm and a residual delayed fluorescence at 430 nm. The excitation spectra for fluorescence and phosphorescence roughly follows 4MU absorption. The phosphorescence lifetime of 4MU is remarkably long, almost 3 s. 4MU excitation and emission phosphorescence anisotropies are low, very close to zero.

The escalating prevalence of hospital-acquired infections poses a critical challenge for healthcare systems worldwide. Effective management requires rapid identification of pathogens and their antibiotic resistance profiles. In this study, we utilized the photoconvertible mEos4b protein, which transitions from green to red fluorescence upon blue light exposure, to distinguish live from dead bacteria. The mEos4b gene was cloned into a prokaryotic vector and expressed in Escherichia coli BL21. The Minimum Inhibitory Concentration (MIC) of the transgenic bacteria was determined for five antibiotics, followed by a post-antibiotic effect assessment over a two-hour exposure period. The optimal photoconversion time for mEos4b was established as 90 s, and confocal microscopy was used to visualize live (green) and dead (red) cells post-exposure. The mEos4b-TR system proved highly specific, accurately distinguishing live and dead bacteria without producing false positives, even in control groups, which is a common issue in commercial live-dead kits. By relying on cellular metabolic activity rather than dyes, this system minimizes nonspecific interactions and contamination, making it more reliable than traditional methods prone to false readings. These results highlight the potential of the mEos4b-TR system as a superior alternative for rapid, precise bacterial viability assessments, particularly in determining antibiotic susceptibility. This preliminary study demonstrates the system’s differentiation of viable and non-viable cells, suggesting its potential application in future studies involving novel antibacterial agents to refine antibiotic sensitivity testing.

Linker-Evolved-Group-Optimized-Lipophosphonoxins (LEGO-LPPO) are small synthetic modular peptidomimetics with promising antimicrobial activity. The LEGO-LPPO mechanism of antibacterial action has been determined to be the depolarization and disruption of bacterial membranes. Their modular nature is advantageous for fine tuning their biological properties. In order to optimize the structure of LEGO-LPPO even further, it is important to understand the interaction of LEGO-LPPO with bacterial membranes at the molecular level. In this work, we present the synthesis of five LEGO-LPPO (designated as 1_naph2-4-G to 5_naph2-4-G) molecules bearing fluorescent naphtylethyl moieties and their usage in the study of LEGO-LPPO behaviour in the membrane. Our goal was to characterize fluorescently labelled LEGO-LPPO under conditions that do not completely disrupt the membrane, mostly in the form of membrane-bound monomers. We observed the intramolecular interactions of hydrophobic modules of 1_naph2-4-G in the buffer by detecting dynamic naphthyl excimers and their disappearance after 1_naph2-4-G bind into the membranes. In the membrane, the molecule 1_naph2-4-G slightly affects the membrane fluidity of DOPG membranes above the phase transition. The naphthyl fluorophore itself has fast and almost unrestricted rotation around ethylene linking groups (r_inf = 0.010), which indicates a considerable chaotropic effect of the hydrophobic modules of 1_naph2-4-G at the given depth of the membrane. 1_naph2-4-G proved to be a useful model for observing the interaction of LEGO-LPPO antibiotics with the phospholipid bilayer enabling us to decipher its effects on membrane state and dynamics; its binding and penetration into the membrane, its structure and the particular depth that it occupies.

Detection of autofluorescence parameters is a useful approach to gain insight into the physiological state of plants and algae, but the effect of reabsorption hinders unambiguous interpretation of in vivo data. The exceptional morphological features of Nitellopsis obtusa made it possible to measure autofluorescence spectra along single internodal cells and estimate relative changes in autofluorescence intensity in selected spectral regions at room temperatures, avoiding the problems associated with thick or optically dense samples. The response of algal cells to controlled white light and DCMU herbicide was analyzed by monitoring changes in peak FL intensity at 680 nm and in F680/F750 ratio. Determining the association between the selected spectral FL parameters revealed an exponential relationship, which provides a quantitative description of photoinduced changes. The ability to discern the effect of DCMU not only in the autofluorescence spectra of dark-adapted cells, but also in the case of light-adapted cells, and even after certain doses of excess light, suggests that the proposed autofluorescence analysis of N. obtusa may be useful for detecting external stressors in the field.

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 ¹S₁–³T₁ and higher-energy ¹S_n–³T_n 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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.