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.

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.

Thermally activated delayed fluorescence (TADF) has recently emerged as one of the most attractive methods for harvesting triplet states in metal-free organic materials for application in organic light emitting diodes (OLEDs). A large number of TADF molecules have been reported in the literature with the purpose of enhancing the efficiency of OLEDs by converting non-emissive triplet states into emissive singlet states. TADF emitters are able to harvest both singlets and triplet states through fluorescence (prompt and delayed), the latter due to the thermally activated reverse intersystem crossing mechanism that allows up-conversion of low energy triplet states to the emissive singlet level. This allows otherwise pure fluorescent OLEDs to overcome their intrinsic limit of 25% internal quantum efficiency (IQE), which is imposed by the 1:3 singlet–triplet ratio arising from the recombination of charges (electrons and holes). TADF based OLEDS with IQEs close to 100% are now routinely fabricated in the green spectral region. There is also significant progress for blue emitters. However, red emitters still show relatively low efficiencies. Despite the significant progress that has been made in recent years, still significant challenges persist to achieve full understanding of the TADF mechanism and improve the stability of these materials. These questions need to be solved in order to fully implement TADF in OLEDs and expand their application to other areas. To date, TADF has been exploited mainly in the field of OLEDs, but applications in other areas, such as sensing and fluorescence microscopies, are envisaged. In this review, the photophysics of TADF molecules is discussed, summarising current methods to characterise these materials and the current understanding of the TADF mechanism in various molecular systems.

The transition dipole moment of organic dyes PM546 and rhodamine 123 is calculated from fluorescence lifetime measurements in solutions of different refractive index. A model proposed by Toptygin et al (2002 J. Phys. Chem. B 106 3724–34) provides a relationship between the radiative rate constant and refractive index of the solvent, and allows the electronic transition dipole moments to be found: it is (7.1  ±  1.1) D for PM546 which matches that found in the literature, and (8.1  ±  0.1) D for rhodamine 123. Toptygin’s model goes further in predicting the shape of the fluorescent dye and here we predict the shape of PM546 and rhodamine 123 to be ellipsoidal.

A well-documented obstacle in the synthesis of functionalized rhodamine dyes is the generation of regioisomers which are difficult to separate. These isomers occur due to the use of unsymmetrical anhydride reagents, which possess two potential points of reactivity where condensation with meta-aminophenols can take place. In this report we describe a method which eliminates this problem by using phthalaldehydic acids as anhydride replacements. These reagents provide only one point of reactivity for the aminophenol, thus allowing direct access to single isomer tetramethylrhodamines and avoiding isomer generation altogether. A range of functionalities are shown to be tolerated at the 5- and 6-position of the dye compounds which are prepared in up to gram quantities using our method. The scope of the method is further demonstrated by the preparation of additional rhodamine family members Rhodamine B and X-Rhodamine.

2-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.

Conventional fragments of fluorescent proteins used in bimolecular fluorescence complementation technique (BiFC), form light-emitting species only when they are kept in close proximity by interacting proteins of interest. By contrast, certain fluorescent protein fragments complement spontaneously, namely those corresponding to the 1st to 10th beta-strands (GFP1-10) and the 11th beta-strand of superfolder GFP (GFP11). They were designed as folding reporters for high throughput expression and structure biology. Besides, for light microscopy, self-associating fluorescent protein fragments constitute a valuable and sometimes unique tool. The GFP11 tag is very advantageous when a full-length fluorescent protein cannot be fused to a protein of interest, namely for live imaging of certain pathogens. Self-associating GFP fragments enable live labelling of specific synapses, visualization of proteins topology and their exposure to particular subcellular compartments. Present review aims to attract attention of scientific community to these tools and to inspire their further development and applications.

In this review, we discuss methods and advancements in fluorescence lifetime imaging microscopy that permit measurements to be performed at faster speed and higher resolution than previously possible. We review fast single-photon timing technologies and the use of parallelized detection schemes to enable high-throughput and high content imaging applications. We appraise different technological implementations of fluorescence lifetime imaging, primarily in the time-domain. We also review combinations of fluorescence lifetime with other imaging modalities to capture multi-dimensional and correlative information from a single sample. Throughout the review, we focus on applications in biomedical research. We conclude with a critical outlook on current challenges and future opportunities in this rapidly developing field.

Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quencher.

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.

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 ¹S ₁– ³T ₁ 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.

The right choice of a fluorescent probe is essential for successful luminescence imaging and sensing and especially concerning in vivo and in vitro applications, the development of new classes have gained more and more attention in the last years. One of the most promising class are upconversion nanoparticles (UCNPs)—inorganic nanocrystals capable to convert near-infrared light in high energy radiation. In this review we will compare UCNPs with other fluorescent probes in terms of (a) the optical properties of the probes, such as their brightness, photostability and excitation wavelength; (b) their chemical properties such as the dispersibility, stability under experimental or physiological conditions, availability of chemical modification strategies for labelling; and (c) the potential toxicity and biocompatibility of the probe. Thereby we want to provide a better understanding of the advantages and drawbacks of UCNPs and address future challenges in the design of the nanocrystals.

Fluorescent sensors benefit from high signal-to-noise and multiple measurement modalities, enabling a multitude of applications and flexibility of design. Semiconductor nanocrystal quantum dots (QDs) are excellent fluorophores for sensors because of their extraordinary optical properties. They have high thermal and photochemical stability compared to organic dyes or fluorescent proteins and are extremely bright due to their large molar cross-sections. In contrast to organic dyes, QD emission profiles are symmetric, with relatively narrow bandwidths. In addition, the size tunability of their emission color, which is a result of quantum confinement, make QDs exceptional emitters with high color purity from the ultra-violet to near infrared wavelength range. The role of QDs in sensors ranges from simple fluorescent tags, as used in immunoassays, to intrinsic sensors that utilize the inherent photophysical response of QDs to fluctuations in temperature, electric field, or ion concentration. In more complex configurations, QDs and biomolecular recognition moieties like antibodies are combined with a third component to modulate the optical signal via energy transfer. QDs can act as donors, acceptors, or both in energy transfer-based sensors using Förster resonance energy transfer (FRET), nanometal surface energy transfer (NSET), or charge or electron transfer. The changes in both spectral response and photoluminescent lifetimes have been successfully harnessed to produce sensitive sensors and multiplexed devices. While technical challenges related to biofunctionalization and the high cost of laboratory-grade fluorimeters have thus far prevented broad implementation of QD-based sensing in clinical or commercial settings, improvements in bioconjugation methods and detection schemes, including using simple consumer devices like cell phone cameras, are lowering the barrier to broad use of more sensitive QD-based devices.

UCNPs have attracted a great deal of attention as near infrared-excited luminescent probes for biomedical applications. UCNPs can provide contrast for in vivo imaging, act as luminescent temperature reporters and excite different molecules to trigger therapeutic processes. While the unique features of UCNPs are well-suited for certain applications, their intrinsic limitations may prevent their general use in preclinical and clinical settings as luminescent probes. In this work, we analyze the role of UCNPs in research in small animal models. The evolution in the field, from the early studies evaluating UCNPs for in vivo fluorescence imaging to the most recent applications, is described, and the advantages and limitations of UCNPs for different applications are discussed. Their adequacy for preclinical research and potential clinical application are also discussed.

In this perspective, we aim to present an overview of some important physical and chemical aspects of lanthanide-doped upconverting nanoparticle research to be considered, from synthesis considerations to bioapplications. To this end, we have reviewed several practical considerations and prepared several straightforward recommendations toward improved cohesion in the field, based on observed trends over the last decade of research.

The fluorescence quantum yield is a measure of the efficiency of photon emission and quantifies the luminescent performance of a given sample. The determination of fluorescence quantum yields, particularly in scattering media, is relevant in the areas of materials science, technology and photonics. It is equally crucial when studying fluorescent bioanalytical probes and biological systems either for medical applications, physiological analyses or the interpretation of optical signals in nature. This type of determination represents a challenge since light scattering introduces an appreciable complexity in the measurements. Hence, the use of experimentally accurate methods and the understanding of their basis and principles is indispensable for obtaining reliable results. In addition, light re-absorption processes are usually very significant in these systems and the experimental quantum yields normally differ from the true quantum yields of the fluorophore. The first purpose of this work is to provide a clear and comprehensive compilation of the various optical methods that can be used for the determination of quantum yields in scattering media. A second purpose is to present the correction models to account for light re-absorption processes, applicable in each case. The advantages and disadvantages of each methodology are comparatively discussed, the difference between experimental and true quantum yield is clarified and it is explained which should be used depending on the case. Several examples previously published in literature are illustrated. The methods presented here are adequate for the study of very diverse samples such as suspensions, solid powders, films, animal tissues and even plant material.

Hospital infections associated with multidrug-resistant (MDR) Pseudomonas aeruginosa are a worldwide public health problem. Efflux systems and biofilm formation are mechanisms related to resistance to carbapenemics. In this study, quantum dots (QDs) were used to evaluate the effect of carbonyl cyanide-3-chlorophenylhydrazone (CCCP), an efflux pump system inhibitor, on biofilm formation and antimicrobial resistance profile of P. aeruginosa strains. For this, QDs were covalently conjugated to meropenem (MPM) and incubated with a P. aeruginosa resistant isolate (P118) or a control sensitive strain (ATCC Pa27853). P118 was also analyzed with conjugates after previous CCCP efflux inhibitor incubation. Fluorescence microscopy images showed that both sensitive and resistant bacteria were efficiently labeled. Nevertheless, P118 isolates presented fluorescent cell agglomerates, suggesting biofilm formation. The addition of the CCCP changed the labeling profile of the resistant isolate, and the absence of agglomerates was observed, indicating no biofilm formation. Genetic assays revealed the presence of MexA and MexE genes encoding channel proteins from efflux pump systems in both resistant and sensitive strains. Disk-diffusion and broth microdilution tests determined drug susceptibility profiles in the presence and absence of CCCP for P118 isolates. We verified that the CCCP efflux system inhibitor may contribute to P. aeruginosa resistant phenotype reduction for some antimicrobials. This study verified the efficiency of QD-MPM conjugates to trigger and study biofilm formation, or its inhibition, before and after CCCP addition. QDs conjugated to antimicrobials can be used as nanotools to investigate multidrug-resistant bacterial strains on biofilm formation.

CsPbBr₃ colloidal quantum dots have been synthesized by hot-injection method showing spherical shape with an average diameter of ~10.5 nm. UV–vis absorption of CsPbBr₃ colloidal quantum dots shows a broad spectrum with an optical bandgap of ~2.3682 eV. The steady-state photoluminescence measurement reveals a narrow emission peak at 2.352 eV with full-width at half maximum of 0.113 eV. Absolute photoluminescence quantum yield of colloidal quantum dots dispersed in poly(methyl methacrylate) was found to be 60 ± 1%. The time-resolved photoluminescence data recorded at 266 nm excitation were well fitted using a mono-exponential curve with a decay time of 25.36 (5) ns. A luminescent solar concentrator was fabricated using colloidal quantum dots in transparent poly(methyl methacrylate) polymer uniformly coated over glass substrate that shows an external optical conversion efficiency of ~5.4% under one sun illumination. The experimental results presented in this manuscript reveals that luminescent solar concentrator prepared using colloidal CsPbBr₃ quantum dots shows absorption in wide spectral range, high absorption coefficient, high photoluminescence quantum yield, high external optical conversion efficiency, and good photostability, thermal stability and long-term stability under ambient conditions and therefore are in many ways superior to the other luminescent materials explored for LSC devices.

There is an increasing need for the development of probes for the detection of hexavalent chromium since it is a known carcinogen, which can cause adverse effects on human health. Metal-organic frameworks (MOFs) have shown successful detection and removal of hazardous substances from aqueous media. This work presents the use of simple organic ligands such as 3-pyridinecarboxaldehyde and trimesic acid with Zn(II) ion to fabricate a new MOF that exhibits sensitive and selective luminescence quenching response towards CrO₄²⁻ and Cr₂O₇²⁻ species in aqueous solution. The MOF showed a detection limit of 0.67 μM (0.078 ppm) as CrO₄²⁻ species and 1.91 μM (0.41 ppm) as Cr₂O₇²⁻ species. Results reveal that the as-synthesized MOF could serve as a good luminescent sensor for CrO₄²⁻ and Cr₂O₇²⁻ species in the contaminated aqueous phase.

The fluorescence quantum yield is a measure of the efficiency of photon emission and quantifies the luminescent performance of a given sample. The determination of fluorescence quantum yields, particularly in scattering media, is relevant in the areas of materials science, technology and photonics. It is equally crucial when studying fluorescent bioanalytical probes and biological systems either for medical applications, physiological analyses or the interpretation of optical signals in nature. This type of determination represents a challenge since light scattering introduces an appreciable complexity in the measurements. Hence, the use of experimentally accurate methods and the understanding of their basis and principles is indispensable for obtaining reliable results. In addition, light re-absorption processes are usually very significant in these systems and the experimental quantum yields normally differ from the true quantum yields of the fluorophore. The first purpose of this work is to provide a clear and comprehensive compilation of the various optical methods that can be used for the determination of quantum yields in scattering media. A second purpose is to present the correction models to account for light re-absorption processes, applicable in each case. The advantages and disadvantages of each methodology are comparatively discussed, the difference between experimental and true quantum yield is clarified and it is explained which should be used depending on the case. Several examples previously published in literature are illustrated. The methods presented here are adequate for the study of very diverse samples such as suspensions, solid powders, films, animal tissues and even plant material.

Intracellular refractive index (RI) is an essential biophysical parameter, which best represents the mass and the distribution of proteins in the cell interior, including high-density accumulations in membraneless organelles. For RI measurements, a number of sophisticated techniques have been developed; however most of the new approaches are either insufficiently sensitive to intracellular variations of proteins distribution or are not compatible with live cell studies. Here, we outline the fluorescence lifetime imaging (FLIM) strategy for high resolution mapping of subcellular RI. We provide an example of our recent studies in which we utilize FLIM for measurements and monitoring of local RI in the major membraneless organelles within live cultured cells.

Photochemical reactions can be designed to convert either irreversibly or reversibly a nonemissive reactant into an emissive product. The irreversible disconnection of a photocleavable group from an emissive chromophore or the reversible interconversion of a photochromic component is generally exploited to implement these operating principles for fluorescence switching. In both instances, the interplay of activating radiation, to convert the nonemissive state into the emissive species, and exciting radiation, to produce fluorescence from the latter, can be exploited to switch fluorescence on in a given area of interest at a precise interval of time. Such a level of spatiotemporal control provides the opportunity to reconstruct sub-diffraction images with resolution at the nanometer level. Indeed, closely-spaced emitters can be switched on under photochemical control at distinct intervals of time and localized independently at the single-molecule level. In combination with appropriate intracellular targeting strategies, some of these photoactivatable fluorophores can be switched and localized inside live cells to permit the visualization of sub-cellular structures with a spatial resolution that would be impossible to achieve with conventional fluorophores. As a result, photoactivatable fluorophores can become invaluable probes for the implementation of super-resolution imaging schemes aimed at the elucidation of the fundamental factors controlling cellular functions at the molecular level.

Although the theoretical foundations of Förster resonance energy transfer (FRET) were laid in the 1940s as part of the quantum physical revolution of the 20th century, it was only in the 1970s that it made its way to biology as a result of the availability of suitable measuring and labeling technologies. Thanks to its ease of application, FRET became widely used for studying molecular associations on the nanometer scale. The development of superresolution techniques at the turn of the millennium promised an unprecedented insight into the structure and function of molecular complexes. Without downplaying the significance of superresolution microscopies this review expresses our view that FRET is still a legitimate tool in the armamentarium of biologists for studying molecular associations since it offers distinct advantages and overcomes certain limitations of superresolution approaches.

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.

The use of organic based fluorophores has been firmly established as a key tool in the biological sciences, with many biological-sensing methods taking advantage of Förster Resonance Energy Transfer (FRET) between different fluorescent organic based dyes following one photon excitation. Nevertheless, the employment of UV-visible absorbing dyes as fluorescent tags and markers typically suffer from several drawbacks including relatively high energy of excitation wavelength, photobleaching and competitive autofluorescence, which often limits their effectiveness and longevity both in vitro and in vivo. As an alternative, lanthanide doped upconverting phosphors (UCP) have emerged as a new class of materials for use in optical imaging and RET sensing; they exhibit high photo- and chemical stability and utilise near infrared excitation. Approaches to sensing a given analyte target employing upconverting phosphors can be achieved by engineering the UCP to operate analogously to fluorescent dyes via Luminescence Resonance Energy Transfer (LRET) and such systems are now becoming central to optically sensing low concentrations of biologically important species and performing distance measurements. Similarly to FRET, the LRET process is distance dependent and requires spectral overlap between the absorption of the acceptor luminophore and the emission of the donor moiety, yet essential measures of the relationship between spectral overlap and the degree of quenching have not yet been established. To address this, we have investigated the Stern-Volmer relationship for a set of six commonly functionalised organic dyes and seven biomolecules that contain key chromophoric co-factors with Gd ₂SO ₄:Yb:Er (PTIR545) and Gd ₂SO ₄:Yb:Tm (PTIR475) UCPs under low power nIR excitation, and found that for the organic dyes a linear relationship between spectral overlap and degree of quenching is observed. However, this linear relationship is observed to break down for all the biomolecules investigated.

We demonstrate that single functionalized silver nanowires form a geometric platform suitable for efficient real-time detection of single photoactive proteins. By collecting series of images using wide-field fluorescence microscopy, events of single protein attachment can be distinguished with the signal to noise ratio further improved by fluorescence enhancement due to plasmon excitations in the nanowires. The enhancement is evidenced by strong shortening of the fluorescence decay of single photoactive proteins conjugated to the silver nanowires.

Facultative intracellular pathogens are able to live inside and outside host cells. It is highly desirable to differentiate their cellular locations for the purposes of fundamental research and clinical applications. In this work, we developed a novel analysis platform that allows users to choose two analysis models: amplitude weighted lifetime ( τ _A) and intensity weighted lifetime ( τ _I) for fluorescence lifetime imaging microscopy (FLIM). We applied these two models to analyse FLIM images of mouse Raw macrophage cells that were infected with bacteria Shigella Sonnei, adherent and invasive E. coli (AIEC) and Lactobacillus. The results show that the fluorescence lifetimes of bacteria depend on their cellular locations. The τ _A model is superior in visually differentiating bacteria that are in extra- and intra-cellular and membrane-bounded locations, whereas the τ _I model show excellent precision. Both models show speedy performances that analysis can be performed within 0.3 s. We also compared the proposed models with a widely used commercial software tool ( τ _C, SPC Image, Becker & Hickl GmbH), showing similar τ _I and τ _C results. The platform also allows users to perform phasor analysis with great flexibility to pinpoint the regions of interest from lifetime images as well as phasor plots. This platform holds the disruptive potential of replacing z-stack imaging for identifying intracellular bacteria.

FRET is both a phenomenon and a spectroscopic technique, capable of measuring one geometric quantity: kappa-squared divided by the sixth power of the donor-acceptor distance. Kappa-squared is often replaced by a constant even though such a replacement may lead to serious errors. Kappaphobia, the fear of kappa or the reluctance to deal with kappa-squared adequately, is a looming presence in the FRET community. Unfortunately, this reluctance, or fear, is often tolerated, and sometimes encouraged. A decrease in kappaphobia will lead to an increase in the impact and success of FRET.

In several cellular systems, the phasor FLIM approach has shown the existence of more than 2 components in the same pixel, a typical example being free and bound NADH. In order to properly quantify the concentrations and the spatial distributions of fluorescence components associated with different molecular species we developed a general method to resolve 3 and 4 components in the same pixel using the phasor approach. The method is based on the law of linear combination of components valid after transformation of the decay curves to phasors for each pixel in the image. In principle, the linear combination rule is valid for an arbitrary number of components. For 3 components we use only the phasor position for the first harmonic, which has a small error, while for 4 components we need the phasor location at higher harmonics that have intrinsically more noise. As a result of the noise in the higher harmonics, caused by limited photon statistics, we are able to use linear algebra to resolve 4 components given the position of the phasors of 4 independent components in mixtures of dyes and 3 components for dyes in cellular systems.

Single-molecule hybridisation of CY3 dye labelled short oligonucleotides to surface immobilised probes was investigated in zero-mode waveguide nanostructures using a modified DNA sequencer. At longer measuring times, we observed changes of the initial hybridisation fluorescence pulse pattern which we attribute to products created by chemical reactions at the nucleobases. The origin is a charge separated state created by a photoinduced electron transfer from nucleobases to the dye followed by secondary reactions with oxygen and water, respectively. The positive charge can migrate through the hybrid resulting in base modifications at distant sites. Static fluorescence spectra were recorded in order to determine the properties of CY3 stacking to different base pairs, and compared to pulse intensities. A characteristic pulse pattern change was assigned to the oxidation of G to 8-oG besides the formation of a number of secondary products that are not yet identified. Further, we present a method to visualise the degree of chemical reactions to gain an overview of ongoing processes. Our study demonstrates that CY3 is able to oxidise nucleobases in ds DNA, and also in ss overhangs. An important finding is the correlation between nucleobase oxidation potential and fluorescence quenching which explains the intensity changes observed in single molecule measurements. The analysis of fluorescence traces provides the opportunity to track complete and coherent reaction sequences enabling to follow the fate of a single molecule over a long period of time, and to observe chemical reactions in real-time. This opens up the opportunity to analyse reaction pathways, to detect new products and short-lived intermediates, and to investigate rare events due to the large number of single molecules observed in parallel.

2-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.

Upconversion nanoparticles have attracted considerable attention as luminescent markers for bioimaging and sensing due to their capability to convert near-infrared (NIR) excitation into visible or NIR luminescence. However, the wavelength of about 970 nm is commonly used for the upconversion luminescence excitation, where the strong absorption of water is observed, which can lead to laser-induced overheating effects. One of the strategies for avoiding such laser-induced heating involves shifting the excitation into shorter wavelengths region. However, the influence of wavelength change on luminescent images quality has not been investigated yet. In this work, we compare wavelengths of 920, 940 and 970 nm for upconversion luminescence excitation in the thickness of biological tissues in terms of detected signal intensity and obtained image quality (contrast and signal-to-background ratio). Studies on biological tissue phantoms with various scattering and absorbing properties were performed to analyze the influence of optical parameters on the depth and contrast of the images obtained under 920–970 nm excitation. It was shown that at the same power the excitation wavelength shift reduces the detected signal intensity and the resulting image contrast. Visualization of biological tissue samples using shorter excitation wavelengths 920 and 940 nm also reduces signal-to-background ratio (S/B) of the obtained images. The S/B of the obtained images amounted to 2, 6 and 8 for 920, 940 and 970 nm, respectively. It was demonstrated that pulse-periodic excitation mode is preferable for obtaining high quality luminescent images of biological tissues deep layers and minimize overheating. Short pulse durations (duty cycle 20%) don’t result in heating even for 1 W cm ⁻² pumping power density and allow obtaining high luminescence intensity, which provides good images quality.

Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (−30)GFP, to a myogenic transcription factor, MyoD. The (−30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (−30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (−30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.