Fluorescence-based Techniques Research Articles

A multi-functional reagent suitable for 1-step rapid DNA intercalation fluorescence-based screening of total bacteria in drinking water

The prerequisites for rapid screening of total bacteria in drinking water are low detection limit and convenience. Inspired by commercial adenosine 5′-triphosphate (ATP) based total bacterial detection kits, we pursued likewise convenience but with much lower detection limit. Existing intercalation fluorescence-based techniques employ multiple reagents to permeate the cell membrane and intercalate dye into the DNA in discrete sequential steps. A simple multi-functional reagent is proposed to do the same within one step. Surfactants (TritonX and SDS), and intercalating dyes (SYBR green, SYBR gold) were examined for their mutual compatibility and augmented with EDTA. Evaluation was performed with Gram negative Escherichia coli K12 (E. coli K12) and Gram positive Bacillus subtilis (B. subtilis) at serial dilution ratios from 10−6 to 10−2. Comparison was made with absorbance (600 nm) measurements and a commercial ATP kit. Using charge integrated photodetection, the proposed 1-step reagent achieved an LOD (1.00 × 10−6, B. subtilis) that is two orders of magnitude lower than that of ATP kit (LOD = 1.06× 10−4). This means it could detect minute quantity of total bacteria that is otherwise undetected by the ATP kit.

Macroscopic and microscopic characterization of fluorescence properties of multiple sweet pepper cultivars (Capsicum annuum L.) using excitation-emission matrix and UV induced fluorescence imaging

Sweet peppers are a popular vegetable with various surface colors, such as green, purple, red, or yellow. To characterize the unique fluorescence properties associated with a broad range of sweet peppers of various colors (14 varieties), a fluorescence spectrofluorometer and imaging were used. The results showed that all cultivars in the experiment had blue fluorescence emissions when excited with light in the UV-A region, while chlorophyll fluorescence could be observed in green peppers. The emitted blue fluorescence originated from the epidermis (cuticle layer). The color distribution of these sweet peppers in the a* and b* color space were compared to the image obtained under white LED light. Yellow and red pepper cultivars have thicker, multiple cuticular wax layers and more distinct maturity stages than other sweet pepper varieties observed. With the establishment of this basic fluorescence database, further applications of fluorescence-based techniques and the unification of evaluation methods for pepper quality will be more easily established.

Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State.

ABSTRACTCryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans.IMPORTANCE Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.

Plasmonic Biosensing for Label-Free Detection of Two Hallmarks of Cancer Cells: Cell-Matrix Interaction and Cell Division.

Two key features of cancer cells are sustained proliferation and invasion, which is preceded by a modification of the adhesion properties to the extracellular matrix. Currently, fluorescence-based techniques are mainly used to detect these processes, including flow cytometry and fluorescence resonance energy transfer (FRET) microscopy. We have previously described a simple, fast and label-free method based on a gold nanohole array biosensor to detect the spectral response of single cells, which is highly dependent on the actin cortex. Here we used this biosensor to study two cellular processes where configuration of the actin cortex plays an essential role: cell cycle and cell–matrix adhesion. Colorectal cancer cells were maintained in culture under different conditions to obtain cells stopped either in G0/G1 (resting cells/cells at the initial steps of cell growth) or G2 (cells undergoing division) phases of the cell cycle. Data from the nanohole array biosensor showed an ability to discriminate between both cell populations. Additionally, cancer cells were monitored with the biosensor during the first 60 min after cells were deposited onto a biosensor coated with fibronectin, an extracellular matrix protein. Spectral changes were detected in the first 20 min and increased over time as the cell–biosensor contact surface increased. Our data show that the nanohole array biosensor provides a label-free and real-time procedure to detect cells undergoing division or changes in cell–matrix interaction in both clinical and research settings.

Highly Selective and Sensitive Ratiometric Detection of Sn2+ Ions Using NIR-Excited Rhodamine-B-Linked Upconversion Nanophosphors

Detection of Sn2+ ions in environmental and biological samples is essential owing to the toxicological risk posed by excess use tin worldwide. Herein, we have designed a nanoprobe involving upconversion nanophosphors linked with a rhodamine-based fluorophore, which is selectively sensitive to the presence of Sn2+ ions. Upon excitation with near-infrared (NIR) light, the green emission of the nanophosphor is reabsorbed by the fluorophore with an efficiency that varies directly with the concentration of the Sn2+ ions. We have explored this NIR-excited fluorescence resonance energy transfer (FRET) process for the quantitative and ratiometric detection of Sn2+ ions in an aqueous phase. We have observed an excellent linear correlation between the ratiometric emission signal variation and the Sn2+ ion concentration in the lower micromolar range. The detection limit of Sn2+ ions observed using our FRET-based nanoprobe is about 10 times lower than that observed using other colorimetric or fluorescence-based techniques. Due to the minimal autofluorescence and great penetration depth of NIR light, this method is ideally suited for the selective and ultrasensitive detection of Sn2+ ions in complex biological or environmental samples.

Nanomaterials Used in Fluorescence Polarization Based Biosensors.

Fluorescence polarization (FP) has been applied in detecting chemicals and biomolecules for early-stage diagnosis, food safety analyses, and environmental monitoring. Compared to organic dyes, inorganic nanomaterials such as quantum dots have special fluorescence properties that can enhance the photostability of FP-based biosensing. In addition, nanomaterials, such as metallic nanoparticles, can be used as signal amplifiers to increase fluorescence polarization. In this review paper, different types of nanomaterials used in in FP-based biosensors have been reviewed. The role of each type of nanomaterial, acting as a fluorescent element and/or the signal amplifier, has been discussed. In addition, the advantages of FP-based biosensing systems have been discussed and compared with other fluorescence-based techniques. The integration of nanomaterials and FP techniques allows biosensors to quickly detect analytes in a sensitive and cost-effective manner and positively impact a variety of different fields including early-stage diagnoses.

Cardiac Myosin Filaments are Maintained by Stochastic Protein Replacement

Myosin and myosin-binding protein C are exquisitely organized into giant filamentous macromolecular complexes within cardiac muscle sarcomeres, yet these proteins must be continually replaced to maintain contractile fidelity. The overall hypothesis that myosin filament structure is dynamic and allows for the stochastic replacement of individual components was tested in vivo, using a combination of mass spectrometry– and fluorescence-based proteomic techniques. Adult mice were fed a diet that marked all newly synthesized proteins with a stable isotope-labeled amino acid. The abundance of unlabeled and labeled proteins was quantified by high-resolution mass spectrometry over an 8-week period. The rates of change in the abundance of these proteins were well described by analytical models in which protein synthesis defined stoichiometry and protein degradation was governed by the stochastic selection of individual molecules. To test whether the whole myosin filaments or the individual components were selected for replacement, cardiac muscle was chemically skinned to remove the cellular membrane and myosin filaments were solubilized with ionic solutions. The composition of the filamentous and soluble fractions was quantified by mass spectrometry, and filament depolymerization was visualized by real-time fluorescence microscopy. Myosin molecules were preferentially extracted from ends of the filaments in the presence of the ionic solutions, and there was only a slight bias in the abundance of unlabeled molecules toward the innermost region on the myosin filaments. These data demonstrate for the first time that the newly synthesized myosin and myosin-binding protein C molecules are randomly mixed into preexisting thick filaments in vivo and the rate of mixing may not be equivalent along the length of the thick filament. These data collectively support a new model of cardiac myosin filament structure, with the filaments being dynamic macromolecular assemblies that allow for replacement of their components, rather than rigid bodies.

Fluorescent behavior of melatonin and related indoleamines in natural deep eutectic solvents

Melatonin is a natural hormone produced by the pineal gland and generated as a special metabolite in plants from tryptophan as a precursor and serotonin as an intermediate. These indoleamines show intrinsic fluorescence properties due to the presence of the indole group in their chemical structure, which made them suitable for the analysis by fluorescence-based techniques. In this research, it is described the first comprehensive study of the fluorescence of melatonin and its indole derivatives in several natural deep eutectic solvents (NADES). As melatonin exhibits solvent-dependent fluorescence characteristics, we have measured the quantum yields of the molecules in NADES and conventional solvents with the purpose of understanding the fluorescent behavior in these solvents that will offer a new alternative to conventional medium. The results showed differential fluorescence behavior of melatonin and its metabolic precursors in NADES that could be explained in terms of each fluorophore structure and potential hydrogen bond interactions with eutectic systems. Finally, the turn-off observed for melatonin in the NADES system based on fructose-citric acid and water could inspire the development of new analytical approaches.

Multi-moded high-index contrast optical waveguide for super-contrast high-resolution label-free microscopy.

The article elucidates the physical mechanism behind the generation of superior-contrast and high-resolution label-free images using an optical waveguide. Imaging is realized by employing a high index contrast multi-moded waveguide as a partially coherent light source. The modes provide near-field illumination of unlabeled samples, thereby repositioning the higher spatial frequencies of the sample into the far-field. These modes coherently scatter off the sample with different phases and are engineered to have random spatial distributions within the integration time of the camera. This mitigates the coherent speckle noise and enhances the contrast (2-10) × as opposed to other imaging techniques. Besides, the coherent scattering of the different modes gives rise to fluctuations in intensity. The technique demonstrated here is named chip-based Evanescent Light Scattering (cELS). The concepts introduced through this work are described mathematically and the high-contrast image generation process using a multi-moded waveguide as the light source is explained. The article then explores the feasibility of utilizing fluctuations in the captured images along with fluorescence-based techniques, like intensity-fluctuation algorithms, to mitigate poor-contrast and diffraction-limited resolution in the coherent imaging regime. Furthermore, a straight waveguide is demonstrated to have limited angular diversity between its multiple modes and therefore, for isotropic sample illumination, a multiple-arms waveguide geometry is used. The concepts introduced are validated experimentally via high-contrast label-free imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs), liposomes, nanobeads and biological cells such as fixed and live HeLa cells.

Fluorescence signal of proteins in birch pollen distorted within its native matrix: Identification of the fluorescence suppressor quercetin-3-O-sophoroside

The properties of biogenic aerosol strongly depend on the particle’s proteinaceous compounds. Proteins from primary biological aerosol particles (PBAPs) can cause allergic reactions in the human respiratory system or act as ice and condensation nuclei in clouds. Consequently, these particles have high impact on human health and climate. The detection of biogenic aerosol is commonly performed with fluorescence-based techniques. However, many PBAPs (i.e., pollen of birch, mugwort, or ragweed) show weak or rather low fluorescence signals in the particular protein region (λex ~ 255–280 nm, λem ~ 280–350 nm). We hypothesize that the fluorescence signal of proteins present in birch pollen is being distorted within its native matrix. In this study, we conducted in vitro quenching experiments and employed UV/Vis spectroscopy, capillary zone electrophoresis (CZE), liquid chromatography (LC), electrospray ionization mass spectrometry (ESI–MS), and multistage MS (MS2 and MS3) to target major components in birch pollen washing water (BPWW) possibly quenching the fluorescence activity of proteins and thus explaining the lack of corresponding protein fluorescent signals. We identified quercetin-3-O-sophoroside (Q3OS, MW 626 g mol−1) to be the main UV/Vis absorbing component in BPWW. Our results point out that Q3OS suppresses the fluorescence of proteins in our samples predominantly due to inner filter effects. In general, when applying fluorescence spectroscopy to analyze and detect PBAPs in the laboratory or the atmosphere, it is important to critically scrutinize the obtained spectra.Graphical abstract

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Impacts of autofluorescence on fluorescence based techniques to study microglia

BackgroundMicroglia, the resident immune cells in the central nervous system, accrue autofluorescent granules inside their cytoplasm throughout their lifespan. In this report, we studied the impacts of autofluorescence on widely used fluorescence-based techniques to study microglia, including flow cytometry, immunofluorescence staining, and live imaging.ResultsThe failed attempt of using fluorescein isothiocyanate (FITC) conjugated antibody to detect lymphocyte-activation gene 3 protein in microglia prompted us to compare the sensitivity of FITC, phycoerythrin (PE) and allophycocyanin (APC) conjugated antibodies to detect surface protein expression in microglia. We found that PE outperformed FITC and APC as the fluorophore conjugated to antibody for flow cytometry by overcoming the interference from microglia autofluorescence. To identify the location and source of microglia autofluorescence, we did confocal imaging and spectral analysis of microglia autofluorescence on fixed brain tissues, revealing that microglia autofluorescence emitted from cytoplasmic granules and displayed a multi-peak emission spectrum. We recommended removing autofluorescence by lipofuscin removing agents when staining intracellular proteins in microglia with the immunofluorescence techniques. On live brain slices, autofluorescent granules reduced the amplitudes of calcium signals in microglial somata derived from GCaMP6s fluorescence and thus needed to be excluded when selecting regions of interest (ROI).ConclusionsIn conclusion, autofluorescence is a critical factor to consider when designing experiments and interpreting results based on fluorescence-based techniques to study microglia.

Label-Free Super-Resolution Imaging Techniques.

Biological and material samples contain nanoscale heterogeneities that are unresolvable with conventional microscopy techniques. Super-resolution fluorescence methods can break the optical diffraction limit to observe these features, but they require samples to be fluorescently labeled. Over the past decade, progress has been made toward developing super-resolution techniques that do not require the use of labels. These label-free techniques span a variety of different approaches, including structured illumination, transient absorption, infrared absorption, and coherent Raman spectroscopies. Many draw inspiration from widely successful fluorescence-based techniques such as stimulated emission depletion (STED) microscopy, photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). In this review, we discuss the progress made in these fields along with the current challenges and prospects in reaching resolutions comparable to those achieved with fluorescence-based methods.

Unfolding the Role of Building Units of MOFs with Mechanistic Insight Towards Selective Metal Ions Detection in Water.

The potential emergence of fluorescence-based techniques has propelled research towards developing probes that can sense trace metal ions specifically. Although luminescent metal-organic frameworks (MOFs) are well suited for this application, the role of building blocks towards detection is not fully understood. In this work, a systematic screening by varying number of Lewis basic (pyridyl-N atoms) sites is carried out in a series of isostructural, robust UiO-67 MOFs, and targeting a model metal ion-Fe3+ . All the three fluorescent MOFs are seen to present quenching response towards Fe3+ ions in water. However, UiO-67@N exhibits highly selective and sensitive response, whereas emission of both UiO-67 and UiO-67@NN is quenched by several metal ions. Detailed experimental and theoretical mechanistic investigation is carried out in addition to demonstration of UiO-67@N being able to sense trace amount of Fe3+ ions in synthetic biological water sample. Further, UiO-67@N based mixed-matrix membrane (MMM) has been prepared and employed to mimic the real time Fe3+ ions detection in water.

Development of Cell Membrane Electrophoresis to Measure the Diffusivity of a Native Transmembrane Protein.

The lateral diffusion of transmembrane proteins in cell membranes is an important process that controls the dynamics and functions of the cell membrane. Several fluorescence-based techniques have been developed to study the diffusivities of transmembrane proteins. However, it is challenging to measure the diffusivity of a transmembrane protein with slow diffusion because of the photobleaching effect caused by long exposure times or multiple exposures to light. In this study, we developed a cell membrane electrophoresis platform to measure diffusivity. We deposited cell membrane vesicles derived from HeLa cells to form supported cell membrane patches. We demonstrated that the electrophoresis platform can be used to drive the movement of not only a lipid probe but also a native transmembrane protein, GLUT1. The movements were halted by the boundaries of the membrane patches and the concentration profiles reached steady states when the diffusion mass flux was balanced with the electrical mass flux. We used the Nernst-Planck equation as the mass balance equation to describe the steady concentration profiles and fitted these equations to our data to obtain the diffusivities. The obtained diffusivities were comparable to those obtained by fluorescence recovery after photobleaching, suggesting the validity of this new method of diffusivity measurement. Only a single snapshot is required for the diffusivity measurement, addressing the problems associated with photobleaching and allowing researchers to measure the diffusivity of transmembrane proteins with slow diffusion.

A Palette of Fluorescent Aβ42 Peptides Labelled at a Range of Surface-Exposed Sites.

Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of A42, involved in Alzheimer’s disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled A42 peptides that can be used to gain understanding of the complex mechanisms of A42 self-assembly and help to develop a more targeted approach to cure the disease.

Development and Characterization of a Fluorescent Ligand for Leukotriene B4 Receptor 2 in Cells and Tissues.

The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), which has been proposed as a promising therapeutic target for diabetic wound healing and gastrointestinal lesions. In this study, the rational design of a fluorescent probe based on the synthetic BLT2 agonist CAY10583 is described. The synthesis of several derivatives of CAY10583 coupled to fluorescein resulted in a traceable ligand suitable for different fluorescence-based techniques. An HTRF-based displacement assay (Tag-lite) on stably transfected CHO-K1 cells was developed to characterize binding properties of diverse BLT2 ligands. Highly specific binding to the BLT2 receptor was demonstrated in staining experiments on mouse skin tissue, and specific modulation of BLT2-induced cAMP signaling provided further evidence for receptor binding and ligand functionality. In conclusion, the fluorescent ligands developed in this study are suitable to investigate the pharmacology of BLT2 receptor ligands in a variety of assay systems.

Intraoperative imaging in pathology-assisted surgery.

The pathological assessment of surgical specimens during surgery can reduce the incidence of positive resection margins, which otherwise can result in additional surgeries or aggressive therapeutic regimens. To improve patient outcomes, intraoperative spectroscopic, fluorescence-based, structural, optoacoustic and radiological imaging techniques are being tested on freshly excised tissue. The specific clinical setting and tumour type largely determine whether endogenous or exogenous contrast is to be detected and whether the tumour specificity of the detected biomarker, image resolution, image-acquisition times or penetration depth are to be prioritized. In this Perspective, we describe current clinical standards for intraoperative tissue analysis and discuss how intraoperative imaging is being implemented. We also discuss potential implementations of intraoperative pathology-assisted surgery for clinical decision-making.

Magnetoelastic Immunosensor via Antibody Immobilization for the Specific Detection of Lysozymes.

Lysozymes in human urine have crucial clinical significance as an indicator of renal tubular and glomerular diseases. Most lysozyme detection methods rely on the enzyme-linked immunosorbent assay (ELISA), which is usually a tedious procedure. Meanwhile, aptamer sensors and fluorescence-based techniques for lysozyme detection have emerged in recent studies. However, these methods are time-consuming and highly complex in operation, and some even require exorbitant reagents and instruments, which restricts real-time clinical monitoring as diagnostic approaches. Therefore, a rapid and low-cost lysozyme detection method with facile preparation is still in demand for modern precision medicine. Herein, we propose a magnetoelastic (ME) immunosensor for lysozyme detection by detecting changes in resonance frequency under a magnetostrictive effect. The detection system is composed of a magnetoelastic chip with an immobilized lysozyme antibody, a solenoid coil, and a vector network analyzer. Since the ME sensor is ultrasensitive to mass change, the frequency offset caused by mass change can be utilized to detect the content of lysozyme. The immunosensor is evaluated to possess superior sensitivity of 138 Hz/μg mL-1 in terms of the resonance frequency shift (RFS). In addition, our sensor displays an outstanding performance in specificity experiments and shows a relatively lower detection limit (1.26 ng/mL) than other conventional lysozyme detection methods (such as ELISA, chemiluminescence assay, fluorescence, and aptamer biosensors).

Using optical interference filters to measure autofluorescence in substrates and coatings

In fluorescence microscopy and related applications, fluorescence from optical elements in the system can be a limiting factor in achieving low background levels. Such “autofluorescence” is considerably weaker than the fluorescence produced by the organic dyes used in fluorescence-based analytical techniques. While spectrally detailed information about autofluorescence is desirable, such information comes at a cost of sensitivity. We present a straightforward approach to making sensitive fluorescence measurements using a simple chopped laser and phase-lock detection scheme. By using high-quality optical filters in the excitation and detection paths, the trade-off between spectral information and ultimate sensitivity is controlled by the choice of filter edge locations and bandwidths. An initial implementation of this approach, using a 405 nm diode laser as an excitation source, achieves a sensitivity of ~1 pW optical power of detected fluorescence over a bandwidth of ~125 nm centered at 505 nm. Data from measurements on glass substrates are presented, and challenges for evaluating autofluorescence from coatings on glass are discussed.