High Background Fluorescence Research Articles

Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging.

Fluorescent light-up aptamers (FLAPs) have emerged as valuable tools to visualize RNAs, but are mostly limited by their poor brightness, low photostability, and high fluorescence background in live cells. Exploiting the avidity concept, here we present two of the brightest FLAPs with the strongest aptamer-dye interaction, high fluorogenicity, and remarkable photostability. They consist of dimeric fluorophore-binding aptamers (biRhoBAST and biSiRA) embedded in an RNA scaffold and their bivalent fluorophore ligands (bivalent tetramethylrhodamine TMR2 and silicon rhodamine SiR2). Red fluorescent biRhoBAST-TMR2 and near-infrared fluorescent biSiRA-SiR2 are orthogonal to each other, facilitating simultaneous visualization of two different RNA species in live cells. One copy of biRhoBAST allows for simple and robust mRNA imaging with strikingly higher signal-to-background ratios than other FLAPs. Moreover, eight biRhoBAST repeats enable single-molecule mRNA imaging and tracking with minimal perturbation of their localization, translation, and degradation, demonstrating the potential of avidity-enhanced FLAPs for imaging RNA dynamics.

Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins13a combined with magnetic beads, chemiluminescence and reverse transcription-recombinase aided amplification for detection of avian influenza a (H7N9) virus.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins (CRISPR-Cas) have promising prospects in the field of nucleic acid molecular diagnostics. However, Clustered Regularly Interspaced Short Palindromic Repeats-based fluorescence detection technology is mainly hindered by proteins with conjugated double bonds and autofluorescence, resulting in high fluorescence background, low sensitivity and incompatible reaction systems, which are not conducive to automatic clinical testing. Chemiluminescence (CL) detection technology has been applied mainly owing to its greatly high sensitivity, as well as low background and rapid response. Therefore, we developed a rapid, ultrasensitive and economical detection system based on Clustered Regularly Interspaced Short Palindromic Repeats-Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins 13a combined with magnetic beads (MBs) and chemiluminescence (CL) (Cas13a-MB-CL) to detect Influenza A (H7N9), an acute respiratory tract infectious disease. The carboxyl functionalized magnetic beads (MBs-COOH) were covalently coupled with aminated RNA probe while the other end of the RNA probe was modified with biotin. Alkaline phosphatase labeled streptavidin (SA-ALP) binds with biotin to form magnetic beads composites. In presence of target RNA, the collateral cleavage activity of Cas13a was activated to degrade the RNA probes on MBs and released Alkaline phosphatase from the composites. The composites were then magnetically separated followed by addition of ALP substrate Disodium 2-chloro-5-{4-methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo (3.3.1.13,7) decan]-4-yl}-1-phenyl phosphate (CDP-star), to generate the chemiluminescence signal. The activity of Associated Proteins 13a and presence of target RNA was quantified by measuring the chemiluminescence intensity. The proposed method accomplished the detection of H7N9 within 30min at 25°C. When combined with Reverse Transcription- Recombinase Aides Amplification (RT-RAA), the low detection limit limit of detection was as low as 19.7fM (3S/N). Our proposed MB-Associated Proteins 13a-chemiluminescence was further evaluated to test H7N9 clinical samples, showing superior sensitivity and specificity.

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains.

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.

Gold Nanoclusters-Based NIR-II Photosensitizers with Catalase-like Activity for Boosted Photodynamic Therapy.

Photodynamic therapy (PDT) under fluorescence imaging as a selective and non-invasive treatment approach has been widely applied for the therapy of cancer and bacterial infections. However, its treatment efficiency is hampered by high background fluorescence in the first near-infrared window (NIR-I, 700–900 nm) and oxygen-dependent photosensitizing activity of traditional photosensitizers. In this work, we employ gold nanoclusters (BSA@Au) with the second near-infrared (NIR-II, 1000–1700 nm) fluorescence and catalase-like activity as alternative photosensitizers to realize highly efficient PDT. The bright NIR-II fluorescence of BSA@Au enables the visualization of PDT for tumor with a high signal-to-background ratio (SBR = 7.3) in 4T1 tumor-bearing mouse models. Furthermore, the catalase-like activity of BSA@Au endows its oxygen self-supplied capability, contributing to a five-fold increase in the survival period of tumor-bearing mice receiving boosted PDT treatment compared to that of the control group. Moreover, we further demonstrate that BSA@Au-based PDT strategy can be applied to treat bacterial infections. Our studies show the great potential of NIR-II BSA@Au as a novel photosensitizer for boosted PDT against cancer and bacterial infections.

Identification of illicit street drugs with swept‐source Raman spectroscopy

AbstractThe identification of the illicit substances in low‐purity seized drugs with Raman spectroscopy is an outstanding problem. The low concentration of the target molecule demands high sensitivity and the presence of impurities produces a strong fluorescence background that makes identification challenging. Although Raman analyzers with 785 and 830 nm excitation wavelengths provide high sensitivity, the longer 1064 nm wavelength produces lower fluorescence. In this work, we demonstrate a Raman spectrometer that simultaneously achieves both high sensitivity and low background fluorescence. We utilize swept‐source Raman spectroscopy in which the spectrometer is replaced by a single high‐collection spectral channel, and the Raman spectrum is swept using a tunable laser. By eliminating the spectrometer and its slit, the optical throughput and sensitivity are improved. Moreover, our swept‐source system requires a single uncooled silicon photodiode that provides higher quantum efficiency at longer wavelengths compared with CCDs. This allowed us to use excitation wavelengths in the 900 nm range to reduce background fluorescence without sacrificing sensitivity. We have demonstrated 6x background fluorescence reduction in colored seized drugs compared with 830 nm excitation. With lower background fluorescence and high sensitivity, we could identify heroin with only 1% purity in a seized sample. Besides forensics and security, the demonstrated swept‐source Raman spectroscopy approach can be a powerful technique in biomedical applications where strong background fluorescence limits detection capabilities.

Comparison of conventional and shifted excitation Raman difference spectroscopy for bacterial identification

AbstractRaman spectroscopy is an emerging tool for fast bacterial identification. However, Raman spectroscopy is depending on suitable preprocessing of the spectra, thereby background removal is a decisive step for conventional Raman spectroscopy. The background has to be estimated, which is challenging especially for high fluorescence backgrounds. Shifted excitation Raman difference spectroscopy (SERDS) eliminates the background through the experimental procedure and holds as promising approach for highly fluorescent samples. Bacterial Raman spectra might be especially complex because these spectra consist of a multitude of overlapping Raman bands from a large multiplicity of biomolecules and only subtitle differences between the species Raman spectra enable the bacterial identification. Here, we investigate the benefits of SERDS compared with conventional Raman spectroscopy specific for the study and identification of bacteria. The comparison utilizes spectra sets of four bacterial species measured with conventional Raman spectroscopy and SERDS and covers three processing approaches for SERDS spectra, for example, the reconstruction with a non‐negative least square algorithm.

Live imaging of RNA and RNA splicing in mammalian cells via the dcas13a-SunTag-BiFC system

Dynamic tracking of the localization of RNA molecules (nucleus and/or cytoplasm) and RNA splicing in living cells plays an important role in understanding their functions. However, a lack of dynamic imaging and high background fluorescence have been reported in the fluorescence in situ hybridization (FISH). Here, we developed a new tool, the dcas13a-SunTag-BiFC system, which fused the dLwacas13a and SunTag systems. dLwacas13a is used as a tracker to target specific RNAs, while SunTag recruits split Venus fluorescent proteins to label targeted RNAs. Our results showed that 4 × NLS-dCas13a-24 × SunTag-BiFC and 2 × NLS- dCas13a-24 × SunTag-BiFC systems can be used for imaging of endogenous RNA foci in the nucleus (Xist) and cytoplasm (Ppib and stress granules) in living cells, respectively. Compared to 12x MS2-MCP system, the dcas13a-SunTag-BiFC system showed a better performance of mRNA foci tracking in live cells. Furthermore, we confirmed the premature termination codon (PTC)-induced exon skipping of Oxt RNA using the dcas13a-SunTag-BiFC and MS2-MCP systems in the nucleus. Thus, the dcas13a-SunTag-BiFC system will facilitate the study of RNA localization in living cells and provide new insights into RNA translocation and splicing.

Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and Label-Free Imaging Techniques.

Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Оптические метки на основе золотых нанозвёзд для ГКР-картирования тканей с использованием красных медицинских лазеров

Star-shaped gold nanoparticles modified with cyanine 5.5 derivatives were studied as optical tags for bioimaging by surface-enhanced Raman scattering (SERS). Obtained tags showed a plasmon resonance band at 640 nm in the absorption spectra which ensures them a maximum efficiency when excited by red lasers. SERS measurements were carried out for the modified particles in the solution phase and after incubation with the PANC-1 cell line. The SERS spectra of the solutions revealed a characteristic signal of cyanine 5.5 amino derivative in the range of 100–1000 cm–1, where there is no high fluorescent background. In cell samples, the SERS spectrum of the dye was recorded from the cytoplasm and was not detected outside the cells. In order to confirm the intracellular localization of the tags, the SERS spectra were also scanned in height. The test on cytotoxicity showed that the studied tags are not toxic in the concentration range from 0.05 to 1 mg/l on atomic gold.

Gold nanostars for SERS mapping of tissues using red medical lasers

Star-shaped gold nanoparticles modified with cyanine 5.5 derivatives were studied as optical tags for bioimaging by surface-enhanced Raman scattering (SERS). Obtained tags showed a plasmon resonance band at 640 nm in the absorption spectra which ensures them a maximum efficiency when excited by red lasers. SERS measurements were carried out for the modified particles in the solution phase and after incubation with the PANC-1 cell line. The SERS spectra of the solutions revealed a characteristic signal of cyanine 5.5 amino derivative in the range of 100-1000 cm-1, where there is no high fluorescent background. In cell samples, the SERS spectrum of the dye was recorded from the cytoplasm and was not detected outside the cells. In order to confirm the intracellular localization of the tags, the SERS spectra were also scanned in height. The test on cytotoxicity showed that the studied tags are not toxic in the concentration range from 0.05 to 1 mg/l on atomic gold. Keywords: surface-enhanced Raman scattering, gold nanostar, cyanine, optical tags, bioimaging

Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

Fungal microparasites (here chytrids) are widely distributed and yet, they are often overlooked in aquatic environments. To facilitate the detection of microparasites, we revisited the applicability of two fungal cell wall markers, Calcofluor White (CFW) and wheat germ agglutinin (WGA), for the direct visualization of chytrid infections on phytoplankton in laboratory-maintained isolates and field-sampled communities. Using a comprehensive set of chytrid–phytoplankton model pathosystems, we verified the staining pattern on diverse morphological structures of chytrids via fluorescence microscopy. Empty sporangia were stained most effectively, followed by encysted zoospores and im-/mature sporangia, while the staining success was more variable for rhizoids, stalks, and resting spores. In a few instances, the staining was unsuccessful (mostly with WGA), presumably due to insufficient cell fixation, gelatinous cell coatings, and multilayered cell walls. CFW and WGA staining could be done in Utermöhl chambers or on polycarbonate filters, but CFW staining on filters seemed less advisable due to high background fluorescence. To visualize chytrids, 1 µg dye mL−1 was sufficient (but 5 µg mL−1 are recommended). Using a dual CFW–WGA staining protocol, we detected multiple, mostly undescribed chytrids in two natural systems (freshwater and coastal), while falsely positive or negative stained cells were well detectable. As a proof-of-concept, we moreover conducted imaging flow cytometry, as a potential high-throughput technology for quantifying chytrid infections. Our guidelines and recommendations are expected to facilitate the detection of chytrid epidemics and to unveil their ecological and economical imprint in natural and engineered aquatic systems.

Intracellular bioorthogonal labeling of glucagon receptor via tetrazine ligation

The third intracellular loop (ICL3) in the cytosolic face of glucagon receptor (GCGR) experiences significant conformational transition during receptor activation. It thus offers an attractive site for the introduction of organic fluorophores in our efforts to construct fluorescence-based GPCR biosensors. Herein, we report our confocal microscopic study of intracellular fluorescent labeling of ICL3 using a bioorthogonal chemistry strategy. Our approach involves the site-specific introduction of a strained alkene amino acid into the ICL3 through genetic code expansion, followed by a highly specific inverse electron-demand Diels-Alder reaction with the fluorescent tetrazine probes. Among the three strained alkene amino acids examined, both SphK and 2′-aTCOK offered successful fluorescent labeling of GCGR ICL3 with the appropriate tetrazine probes. At the same time, 4′-TCOK gave high background fluorescence due to its intracellular retention. The fluorescent tetrazine probes were designed following a computational model for background-free intracellular fluorescent labeling; however, their performance varied significantly in live-cell imaging as the strong non-specific signals interfered with the specific ones. Among all GCGR ICL3 mutants bearing a strained alkene, the H339SphK/2′-aTCOK mutants provided the best reaction partners for the BODIPY-Tz1/4 reagents in the bioorthogonal labeling reactions. The results from this study highlight the challenges in identifying bioorthogonal reactant pairs suitable for intracellular labeling of low-abundance receptors in live-cell imaging studies.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

ABSTRACTFluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

Optimization of PTFE Coating on PDMS Surfaces for Inhibition of Hydrophobic Molecule Absorption for Increased Optical Detection Sensitivity.

Polydimethylsiloxane (PDMS) is a polymer widely used for fabrication and prototyping of microfluidic chips. The porous matrix structure of PDMS allows small hydrophobic molecules including some fluorescent dyes to be readily absorbed to PDMS and results in high fluorescent background signals, thereby significantly decreasing the optical detection sensitivity. This makes it challenging to accurately detect the fluorescent signals from samples using PDMS devices. Here, we have utilized polytetrafluoroethylene (PTFE) to inhibit absorption of hydrophobic small molecules on PDMS. Nile red was used to analyze the effectiveness of the inhibition and the absorbed fluorescence intensities for 3% and 6% PTFE coating (7.7 ± 1.0 and 6.6 ± 0.2) was twofold lower compared to 1% and 2% PTFE coating results (17.2 ± 0.5 and 15.4 ± 0.5). When compared to the control (55.3 ± 1.6), it was sevenfold lower in background fluorescent intensity. Furthermore, we validated the optimized PTFE coating condition using a PDMS bioreactor capable of locally stimulating cells during culture to quantitatively analyze the lipid production using Chlamydomonas reinhardtii CC-125. Three percent PTFE coating was selected as the optimal concentration as there was no significant difference between 3% and 6% PTFE coating. Intracellular lipid contents of the cells were successfully stained with Nile Red inside the bioreactor and 3% PTFE coating successfully minimized the background fluorescence noise, allowing strong optical lipid signal to be detected within the PDMS bioreactor comparable to that of off-chip, less than 1% difference.

Intraoperative Use of Indocyanine Green Fluorescence in Hepatobiliary and Pancreatic Cancer Resections: Current State and Considerations

Introduction: Surgery for hepatobiliary and pancreatic malignancies often has unfavourable outcomes due to incomplete resection, with positive histopathological margins and locoregional metastases. Intraoperative use of indocyanine green fluorescence (ICG) with near-red infrared light can improve tumor detectability and achieve R0 status. Methods: Review of current literature. Results: ICG fluorescence and bile excretion allows for real-time visualisation of malignant tissues and assessment of resection margins. In liver surgery it can identify subcapsular lesions, a property especially useful in minimally invasive surgery where there is loss of tactile feedback and direct visual inspection. It can also delineate hepatic segmental anatomy for anatomical resections. Hepatocellular carcinomas tend to uniformly uptake ICG, whereas primary or metastatic adenocarcinomas like cholangiocarcinomas and colorectal liver metastases as well as poorly differentiated hepatocellular carcinomas show rim enhancement. In pancreatic cancer, fluorescence of the resection margin has been shown to correspond to presence of malignancy on histopathological assessement. ICG can also help to detect micrometastases and extrahepatic spread and distinguish between scar tissue and disease following neoadjuvant chemoradiotherapy. However the technique has limitations: wide variability of method and dose of administration, no visualisation of deep seated lesions, high background fluorescence, false positives up to 40% requiring additional verification by other modalities like intraoperative ultrasound or frozen section for newly detected lesions. Conclusion: ICG fluorescence can offer increased detectability of malignancy and improve post-operative outcomes in hepatobiliary and pancreatic resections. However there are limitations such as superficial depth of detection and low specificity which need improvement to achieve maximum benefit.

A CRISPR/Cas12a Based Universal Lateral Flow Biosensor for the Sensitive and Specific Detection of African Swine-Fever Viruses in Whole Blood.

Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR–Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10−15 M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.

Ocena rearanżacji genu ROS1 przy pomocy fluorescencyjnej hybrydyzacji in situ w niedrobnokomórkowym raku płuca

Introduction. The rearrangement of the gene encoding ROS protooncogene (ROS1) is observed in a very small percentage (1–2%) of patients with non-small cell lung cancer (NSCLC). The clinical characteristics of ROS1-positive patients are similar to those observed in the group of patients with ALK gene rearrangement. Detection of ROS1 gene rearrangement is an extremely important predictive factor enabling the use of crizotinib in the 1st line of NSCLC patients with stage IIIB or IV. Due to the addition of crizotinib to the list of reimbursed drugs from January 2019, the analysis of this genetic change should be part of a molecular tests panel performed in patients with locally advanced and advanced NSCLC in the qualification for molecularly targeted treatment. Aim of the study. Analysis of ROS1 gene rearrangement incidence among NSCLC patients in stage IIIB or IV qualified for molecularly targeted therapies. Presentation of methodological difficulties with fluorescent in situ hybridization (FISH) technique which is used to detect ROS1 genetic abnormality. Materials and methods. The analysis of ROS1 gene rearrangement was carried out using fluorescent in situ hybridization technique in tissue samples taken from 573 NSCLC patients of non-squamous cell type during routine pathomorphological diagnostics. Results. The material obtained from the tumor was fixed in formalin and archived in paraffin. Histological material was obtained from 408 patients, and 165 — cytological (cytoblock). A reliable (diagnostic) result of the ROS1 gene rearrangement was obtained in 439 patients (76.61%). The main difficulties for ROS1 gene analysis were low number of cancer cells, as well as high background fluorescence interference and fragmentation of cell nuclei. ROS1 gene rearrangement was detected in 9 patients with adenocarcinoma (1.57% among all patients), including 5 men and 4 women. In 19 patients, other abnormalities regarding the ROS1 gene were observed, primarily the polysomy of the examined ROS1 gene fragment (3.32%). Polysomy did not coexist with the ROS1 rearrangement. Conclusion. Fluorescent in situ hybridization is a useful tool in detecting ROS1 gene rearrangement. The test can be performed in both histological and cytological material (cytoblock). However, the correct fixation of the material and the appropriate number of tumor cells in the tested samples is extremely important for obtaining a reliable result.

Raman scattering detection of high explosives on human hair

High explosives (HE) represent a high risk to the safety and health of the general population. Therefore, there is an ongoing demand for methods of analysis with limits of detection at trace levels for these hazardous chemicals. Since human hair is one of the main physical attributes of our bodies, the interaction of hair surfaces with HE can be of critical importance in forensic and security applications. Noninvasive in situ approaches such as spectroscopic methods are preferred for elucidating these interactions. Among the spectroscopy-based methodologies, those based on Raman scattering are often favored because the sharpness of the vibrational signals facilitates the precise location of the peak maxima and enables more precise determination of the band shifts due to intermolecular interactions. This preliminary study is based on the detection of the HEs 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN) on human hair strands by RS. Raman spectral libraries obtained with a 660-nm laser excitation line were generated for black, bleached, and natural gray hair using direct deposition of PETN, TNT, and RDX. Results obtained were confirmed using portable systems operating at 785 and 1064 nm. Spectral data were preprocessed to correct a high fluorescence background exhibited by the samples due to the indole groups and melanin present in hair. Despite the high fluorescence levels that characterized all the samples, the vibrational signatures that identify the presence of the HEs studied could be detected once the optimum acquisition parameters were established. Among the three hair types studied, gray hair was the best substrate to observe the vibrations of HE crystals on hair. A method limit of detection of 2 ± 1 ng was estimated from image analysis of the HE crystals observed and confirmed as ∼3 ± 1 ng using spectroscopic analysis.

A novel DSN-based fluorescence assay for MicroRNA-133a detection and its application for LVH diagnosis in maintenance hemodialysis patients.

Left ventricular hypertrophy (LVH) is the most powerful predictor of cardiovascular mortality in maintenance hemodialysis (MHD) patients. Circulating microRNA‐133a (miR‐133a) was reported to be a potential biomarker for LVH in MHD patients. The aim of this experiment is to establish a novel DSN (duplex‐specific‐nuclease)‐based fluorescence assay for the ultrasensitive detection of miR‐133a and investigate its application for LVH diagnosis in MHD patients. The results indicate DSN enzyme combined with ultrathin metallic MoS2 nanosheets presents high sensitivity, specificity, and low fluorescence background for miR‐133a detection. Then, circulating miR‐133a levels in plasma from 40 MHD patients and 20 healthy controls are analyzed by such assay. The levels of miR‐133a are down‐regulated in MHD patients with LVH compared to MHD patients without LVH and healthy controls, and the ROC (receiver operating characteristic) curve shows strong separation between MHD with LVH patients and MHD without LVH patients. Furthermore, the liner regression analysis shows negative correlation of miR‐133a level and interventricular septum thickness (IVS) as well as left ventricular mass index (LVMI), the indicators of LVH. Therefore, our findings reveal DSN‐based fluorescence assay for miR‐133a is suitable for LVH diagnosis in MHD patients.

Feasibility of integrated high‐wavenumber Raman imaging and fingerprint Raman spectroscopy for fast margin assessment in breast cancer surgery

AbstractIntraoperative assessment of surgical margins remains one of the main challenges in cancer surgery. Raman spectroscopy can detect cancer cells with high accuracy, but it is time‐consuming. In this paper, we investigated a selective‐sampling Raman spectroscopy approach, based on high wavenumber (HW) Raman imaging (spectral range 2,500–3,500 cm−1) and fingerprint Raman spectroscopy (spectral range 600–1,800 cm−1), to reduce the overall tissue analysis time while maintaining high diagnostic accuracy. HW Raman mapping was used as a first step to identify the adipose tissue regions based on the C–H stretching bands at 2,700–2,950 cm−1. As residual tumors are typically found in nonadipose tissue, an algorithm was developed to allocate sampling points for fingerprint Raman spectroscopy at locations corresponding to low intensity in the HW‐Raman maps. Preliminary results show that HW‐Raman imaging based on a 671 nm laser is effective and fast for mapping of adipose tissue in breast resections, with typical imaging times of 2 min for tissue areas as large as 2 × 2 cm2 areas. Albeit the remaining high fluorescence background in the fingerprint region prevents the use of single 671‐nm laser, the HW Raman imaging can be still exploited in combination with 785‐nm excitation Raman spectroscopy for identifying residual tumor. Although this study demonstrates the feasibility of this approach, further improvements, such as using single element detectors for HW Raman imaging, are required to increase the analysis speed further towards intraoperative use in the routine clinical setting.