Background Fluorescence Research Articles

Afterglow Nanoprobe-Enabled Quantitative Lateral Flow Immunoassay by a Palm-Size Device for Household Healthcare.

Lateral flow immunoassay (LFIA), a classical point-of-care testing (POCT) technique, plays an important role in disease screening and healthcare monitoring. However, traditional LFIA is either designed for qualitative analysis or requires expensive equipment for quantification, limiting its use in household diagnosis. In this study, we proposed a new generation of LFIA for household health monitoring by using ultralong organic phosphorescence (UOP) nanomaterials as afterglow nanoprobes with a self-developed palm-size sensing device. The UOP nanoprobes exhibit a phosphorescence signal with a second-level lifetime, which completely avoids the interference from excitation light and biological background fluorescence. Therefore, an ultraminiaturized and low-cost UOP nanosensor was successfully designed by eliminating the complex optical path and filtering systems. We chose an inflammatory factor, C-reactive protein (CRP), for household POCT validation. The whole analysis was completed within 9 min. A limit of detection (LOD) of 0.54 ng/mL of CRP antigen was achieved with high stability and good specificity, which is comparable to laboratory instruments and fully satisfying the clinical diagnosis requirement.

A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane.

Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take Vibrio parahaemolyticus DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.

Solvatochromic Buffering Fluorescent Probe Resolves the Lipid Transport and Morphological Changes during Lipid Droplet Fusion by Super-Resolution Imaging.

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF, a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.

Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.

N-Aryl-DABCO Salts as an Unprecedented Sensing Platform for the Detection of Thiols and Selenols.

Quaternary N-aryl-DABCO salts were introduced for the first time as a highly selective sensing platform for thiols and selenols. By employing this platform, a highly sensitive coumarin based "off-on" fluorescent probe was designed and synthesized. The probe possesses a good solubility in water, low background fluorescence, and, most importantly, demonstrates high selectivity to aryl thiols and selenols over their aliphatic counterparts and other common nucleophiles. A dramatic increase in fluorescence intensity is achieved through the selective cleavage of the quaternized DABCO-ring, yielding a piperazine derivatives with a high fluorescence quantum yield (~72 %). Moreover, stability of the probe to the most used reducing agents DTT and TCEP was demonstrated. The limits of detection for p-thiocresol and phenyl selenide were evaluated to be 22 nM and 6 nM, respectively.

Characterization and Formation Mechanism of Ag2MoO4 Crystals via Precipitation Method: Influence of Experimental Parameters and Crystal Morphology

Ag2MoO4 crystals were prepared by a precipitation method by mixing parent solutions of silver nitrate and sodium molybdate. The effects of experimental parameters such as temperature, concentration, and pH were studied. The samples were found to be crystalline, pure, and monophasic in all cases, except in the case of very low pH. The precipitation leads to the formation of the cubic phase β and no secondary phase is present within the limits of detection of XRD and Raman spectroscopy. However, in some cases, at high temperature and high pH, silver oxide nanoparticles form on the surfaces of the crystals, which create a fluorescence background in the Raman spectra, which is otherwise absent. Analyses with the electron microscope have highlighted that the most common crystalline shape is octahedral, which is the most prevalent at low temperatures and pH around 7, but others are also possible, in particular at high temperatures. No growth occurs after precipitation, so crystals with different appearances form at the same time.

Fluorescence guided surgery imaging systems for breast cancer identification: a systematic review.

Breast-conserving surgery (BCS) is limited by high rates of positive margins and re-operative interventions. Fluorescence-guided surgery seeks to detect the entire lesion in real time, thus guiding the surgeons to remove all the tumor at the index procedure. Our aim was to identify the optimal combination of a camera system and fluorophore for fluorescence-guided BCS. A systematic review of medical databases using the terms "fluorescence," "breast cancer," "surgery," and "fluorescence imaging" was performed. Cameras were compared using the ratio between the fluorescent signal from the tumor compared to background fluorescence, as well as diagnostic accuracy measures, such as sensitivity, specificity, and positive predictive value. Twenty-one studies identified 14 camera systems using nine different fluorophores. Twelve cameras worked in the infrared spectrum. Ten studies reported on the difference in strength of the fluorescence signal between cancer and normal tissue, with results ranging from 1.72 to 4.7. In addition, nine studies reported on whether any tumor remained in the resection cavity (5.4% to 32.5%). To date, only three studies used the fluorescent signal for guidance during real BCS. Diagnostic accuracy ranged from 63% to 98% sensitivity, 32% to 97% specificity, and 75% to 100% positive predictive value. In this systematic review, all the studies reported a clinically significant difference in signal between the tumor and normal tissue using various camera/fluorophore combinations. However, given the heterogeneity in protocols, including camera setup, fluorophore studied, data acquisition, and reporting structure, it was impossible to determine the optimal camera and fluorophore combination for use in BCS. It would be beneficial to develop a standardized reporting structure using similar metrics to provide necessary data for a comparison between camera systems.

Intelligent detection of tetracycline by a rare earth multicolor fluorescent probe based on guanosine-5′-monophosphate

Long-term discharge of tetracycline (TC) in the environment exerts selective pressure on the bacteria, increasing resistance in sensitive bacteria and posing a serious threat to public health. Therefore, it is crucial to develop real-time detection of tetracycline. In this study, we reported a novel rare-earth fluorescent nanosensor FITC-GMP-Eu, based on the formation of guanosine-5′-monophosphate disodium salt (GMP) and fluorescein isothiocyanate (FITC), which enabled the real-time identification of tetracycline in the environment. When there was no TC in the environmental sample, the FITC-GMP-Eu nanosensor exhibited the green fluorescence of FITC due to the fluorescence quenching effect of water molecules on Eu3+. When the environmental samples contained TC, the red fluorescence of Eu3+ could be sensitized due to the diketone structure in the tetracycline molecule, which enabled the naked-eye visualized identification of TC. The nanosensor had high sensitivity (detection limit of 19 nM) and a wide detection range (0–30 μM) for TC. At the same time, the sensor was characterized by high visualization and anti-interference ability due to the strong fluorescence of both FITC and Eu complexes. In order to improve the practicality of the detection, this study also prepared a paper-based and sodium alginate gelated fluorescence detection platform with low background fluorescence. Combined with the common color analysis and RGB extraction procedures in mobile phones, real-time and on-site identification of TC on the surface of various fruit samples can be achieved.

Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires.

The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.

Coherent Electron-Vibron Interactions in Surface-Enhanced Raman Scattering (SERS).

In this Letter we identify coherent electron-vibron interactions between near-resonant and nonresonant electronic levels that contribute beyond standard optomechanical models for off-resonant or resonance surface-enhanced Raman scattering (SERS). By developing an open-system quantum model using first molecular interaction principles, we show how the Raman interference of both resonant and nonresonant contributions can provide several orders of magnitude modifications of the SERS peaks with respect to former optomechanical models and over the fluorescence backgrounds. This cooperative optomechanical mechanism allows for generating an enhancement of nonclassical photon pair correlations between Stokes and anti-Stokes photons, which can be detected by photon-counting measurements. Our results demonstrate Raman enhancements and suppressions of coherent nature that significantly impact the standard estimations of the optomechanical contribution from SERS spectra and their quantum mechanical observable effects.

An ultrasensitive smartphone-assisted bicolor-ratiometric fluorescence sensing platform based on a “noise purifier” for point-of-care testing of pathogenic bacteria in food

Non-specific binding in fluorescence resonance energy transfer (FRET) remains a challenge in foodborne pathogen detection, resulting in interference of high background signals. Herein, we innovatively reported a dual-mode FRET sensor based on a “noise purifier” for the ultrasensitive quantification of Escherichia coli O157:H7 in food. An efficient FRET system was constructed with polymyxin B-modified nitrogen-sulfur co-doped graphene quantum dots (N, S-GQDs@PMB) as donors and aptamer-modified yellow carbon dots (Y-CDs@Apt) as acceptors. Magnetic multi-walled carbon nanotubes (Fe@MWCNTs) were employed as a “noise purifier” to reduce the interference of the fluorescence background. Under the background purification mode, the sensitivity of the dual-mode signals of the FRET sensor has increased by an order of magnitude. Additionally, smartphone-assisted colorimetric analysis enabled point-of-care detection of E. coli O157:H7 in real samples. The developed sensing platform based on a “noise purifier” provides a promising method for ultrasensitive on-site testing of trace pathogenic bacteria in various foodstuffs.

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

Super-Resolution Diamond Magnetic Microscopy of Superparamagnetic Nanoparticles.

Scanning-probe and wide-field magnetic microscopes based on nitrogen-vacancy (NV) centers in diamond have enabled advances in the study of biology and materials, but each method has drawbacks. Here, we implement an alternative method for nanoscale magnetic microscopy based on optical control of the charge state of NV centers in a dense layer near the diamond surface. By combining a donut-beam super-resolution technique with optically detected magnetic resonance spectroscopy, we imaged the magnetic fields produced by single 30 nm iron-oxide nanoparticles. The magnetic microscope has a lateral spatial resolution of ∼100 nm, and it resolves the individual magnetic dipole features from clusters of nanoparticles with interparticle spacings down to ∼190 nm. The magnetic feature amplitudes are more than an order of magnitude larger than those obtained by confocal magnetic microscopy due to the narrower optical point-spread function and the shallow depth of NV centers. We analyze the magnetic nanoparticle images and sensitivity as a function of the microscope's spatial resolution and show that the signal-to-noise ratio for nanoparticle detection does not degrade as the spatial resolution improves. We identify sources of background fluorescence that limit the present performance, including diamond second-order Raman emission and imperfect NV charge state control. Our method, which uses <10 mW laser power and can be parallelized by patterned illumination, introduces a promising format for nanoscale magnetic imaging.

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer.

Real-time quaking-induced conversion (RT-QuIC) assays have become a common tool to detect chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for methods to optimize the RT-QuIC assay. In this study, receiver operating characteristic (ROC) analysis was applied to optimize the assay duration for CWD screening in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (Tstdev) and a max-point ratio (maximum/background fluorescence) threshold (TMPR) to determine CWD positivity. The optimal assay duration was 33 h for obex and 30 h for RLN based on Tstdev, and 29 h for obex and 32 h for RLN based on TMPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. At a replicate level, using the optimized assay durations with TStdev and TMPR, the level of agreement with enzyme-linked immunosorbent assay (ELISA) was significantly higher (p < 0.05) than that when using a 40 h assay duration. These findings demonstrate that the optimization of assay duration via a ROC analysis can improve RT-QuIC assays for screening CWD in white-tailed deer.

Tracing endogenous proteins in living cells through electrotransfer of mRNA encoding chromobodies.

Chromobodies made of nanobodies fused to fluorescent proteins are powerful tools for targeting and tracing intracellular proteins in living cells. Typically, this is achieved by transfecting plasmids encoding the chromobodies. However, an excess of unbound chromobody relative to the endogenous antigen can result in high background fluorescence in live cell imaging. Here, we overcome this problem by using mRNA encoding chromobodies. Our approach allows one to precisely control the amount of chromobody expressed inside the cell by adjusting the amount of transfected mRNA. To challenge our method, we evaluate three chromobodies targeting intracellular proteins of different abundance and cellular localization, namely lamin A/C, Dnmt1 and actin. We demonstrate that the expression of chromobodies in living cells by transfection of tuned amounts of the corresponding mRNAs allows the accurate tracking of their cellular targets by time-lapse fluorescence microscopy.

Reaction-Initiated Single-Molecule Tracking of Mass Transfer in Core-Shell Mesoporous Silica Particles.

Understanding the host-guest interactions in porous materials is of great importance in the field of separation science. Probing it at the single-molecule level uncovers the inter- and intraparticle inhomogeneity and establishes structure-property relationships for guiding the design of porous materials for better separation performance. In this work, we investigated the dynamics of host-guest interactions in core-shell mesoporous silica particles under in situ conditions by using a fluorogenic reaction-initiated single-molecule tracking (riSMT) approach. Taking advantage of the low fluorescence background, three-dimensional (3D) tracking of the dynamics of the molecules inside the mesoporous silica pore was achieved with high spatial precision. Compared to the commonly used two-dimensional (2D) tracking method, the 3D tracking results show that the diffusion coefficients of the molecules are three times larger on average. Using riSMT, we quantitatively analyzed the mass transfer of probe molecules in the mesoporous silica pore, including the fraction of adsorption versus diffusion, diffusion coefficients, and residence time. Large interparticle inhomogeneity was revealed and is expected to contribute to the peak broadening for separation application at the ensemble level. We further investigated the impact of electrostatic interaction on the mass transfer of molecules in the mesoporous silica pore and discovered that the primary effect is on the fraction rather than their diffusion rates of resorufin molecules undergoing diffusion.

Lanthanide-Complex-Enhanced Bioorthogonal Branched DNA Amplification.

Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.

Unveiling angular sweep total fluorescence Spectroscopy:A novel multidimensional technique for analysis of complex multi-fluorophoric systems

Analyzing multi-fluorophoric real systems without pre-processing is challenging, often addressed with unconventional fluorescence techniques and chemometrics. In this context, we introduce a novel addition - ‘Angular Sweep Total Fluorescence Spectroscopy (ASTFS),' to the arsenal of multidimensional steady-state fluorescence spectroscopic techniques. ASTFS utilizes a series of variable angle synchronous fluorescence spectra, strategically covering the fluorescence region between the first and second-order Rayleigh scattering ridges. The plot features a minimal data matrix size, avoids Rayleigh scattering signals, and incurs no blind regions. The study delves into the instrumental configurations for spectral acquisition, highlights the enhanced spectral resolution due to the band-narrowing effect, and discusses other notable features of the ASTFS plot. Further, this technique is reported to be effective in analyzing analytes in complex systems with strong background fluorescence, such as milk. The antibiotic— Norfloxacin is quantified via minimal pre-processing in milk samples and yields excellent analytical figures of merit.

Analytical method for stable background reduction for Raman spectra of carbon‐containing meteorite and terrestrial samples suffering from intense fluorescence

AbstractChemical states of carbon in terrestrial (meta) sediments and carbonaceous chondrites gather attention as a geothermometer. As a nondestructive analytical method, Raman spectroscopy has been widely used to study their electronic properties, crystallinity, and structural defects through so‐called D and G bands. For the analysis of Raman spectra, a common problem is coexistence of a fluorescence background, which should be subtracted prior to the peak‐fitting analysis. However, we recently faced a problem that the band shape noticeably changed depending on the background function assumed although the background seemed to be well subtracted at a first glance regardless of the choice of the background function. For the application of the Raman spectroscopy as a geothermometer, a standard background subtraction method must be established to suppress the arbitrariness. In the present study, Raman spectra of seven carbon‐containing natural samples, whose background intensities were significantly different, were measured, and their background shape was evaluated by first‐, second‐, and third‐order polynomials. The results indicated that the third‐order polynomial was necessary and sufficient as a standard background function. Importantly, although lower order polynomials seem to successfully fit the background at a first glance, they falsely caused dispersion of the shoulder band shape.

Evaluation of selected tropical marine microalgal cultures for use in biophotovoltaic platforms

In this study, the bioelectrical power generation potential of four tropical marine microalgal strains native to Malaysia was investigated using BPV platforms. Chlorella UMACC 258 produced the highest power density (0.108 mW m−2), followed by Halamphora subtropica UMACC 370 (0.090 mW m−2), Synechococcus UMACC 371 (0.065 mW m−2) and Parachlorella UMACC 245 (0.017 mW m−2). The chlorophyll-a (chl-a) content was examined to have a linear positive relationship with the power density (p < 0.05). The photosynthetic performance of strains was studied using the pulse-amplitude modulation (PAM) fluorometer; parameters measured include the following: maximum quantum efficiency (Fv/Fm), alpha (α), maximum relative electron transport rate (rETRmax), photo-adaptive index (Ek) and non-photochemical quenching (NPQ). The Fv/Fm values of all strains, except Synechococcus UMACC 371, ranged between 0.37 and 0.50 during exponential and stationary growth phases, suggesting their general health during those periods. The low Fv/Fm value of Synechococcus UMACC 371 was possibly caused by the presence of background fluorescence from phycobilisomes or phycobiliproteins. Electrochemical studies via cyclic voltammetry (CV) suggest the presence of electrochemically active proteins on the cellular surface of strains on the carbon anode of the BPV platform, while morphological studies via field emission scanning electron microscope (FESEM) imaging verify the biocompatibility of the biofilms on the carbon anode.Key points• Maximum power output of 0.108 mW m−2 is recorded by Chlorella UMACC 258• There is a positive correlation between chl-a content and power output• Proven biocompatibility between biofilms and carbon anode sans exogenous mediators