Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique.

Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag-Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag-Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.

High-Throughput All-Optical Determination of Nanorod Size and Orientation.

As a single-particle characterization technique, optical microscopy has transformed our understanding of structure-function relationships of plasmonic nanoparticles, but the need for ex-situ-correlated electron microscopy to obtain structural information handicaps an otherwise exceptional high-throughput technique. Here, we present an all-optical alternative to electron microscopy to accurately and quickly extract structural information about single gold nanorods (Au NRs) using calcite-assisted localization and kinetics (CLocK) microscopy. Color CLocK images of single Au NRs allow scattering from the longitudinal and transverse plasmon modes to be imaged simultaneously, encoding spectral data in CLocK images that can then be extracted to obtain Au NR size and orientation. Moreover, through the use of convolutional neural networks, Au NR length, width, and aspect ratio can be predicted directly from color CLocK images within ∼10% of the true value measured by electron microscopy.

Unusual dark-field hyperspectral and confocal Raman microscopy features of a nanoporous gold electrode coated with porphyrazine complex.

Nanoporous gold electrodes are of great interest in electroanalytical chemistry, because of their unusual activity and large surface area. The electrochemical activity can be further improved by coating with molecular catalysts such as the tetraruthenated cobalt-tetrapyridylporphyrazines investigated in this work. The plasmonic enhancement of the scattered light at the nanoholes and borders modifies the electrode's optical characteristics, improving the transmission through the surface-enhanced Raman scattering (SERS) effect. When monitored by hyperspectral dark-field and confocal Raman microscopy, this effect allows probing of the porphyrazine species at the plasmonic nanholes, improving the understanding of the chemically modified gold electrodes.

Noninvasive assessment of human microvascular function in health and disease using incident dark-field microscopy.

Reduced peripheral microvascular reactivity is associated with an increased risk for major adverse cardiac events (MACEs). Tools for noninvasive assessment of peripheral microvascular function are limited, and existing technology is poorly validated in both healthy populations and patients with cardiovascular disease (CVD). Here, we used a handheld incident dark-field imaging tool (CytoCam) to test the hypothesis that, compared with healthy individuals (no risk factors for CVD), subjects formally diagnosed with coronary artery disease (CAD) or those with ≥2 risk factors for CAD (at risk) would exhibit impaired peripheral microvascular reactivity. A total of 17 participants (11 healthy, 6 at risk) were included in this pilot study. CytoCam was used to measure sublingual microvascular total vessel density (TVD), perfused vessel density (PVD), and microvascular flow index (MFI) in response to the topical application of acetylcholine (ACh) and sublingual administration of nitroglycerin (NTG). Baseline MFI and PVD were significantly reduced in the at-risk cohort compared with healthy individuals. Surprisingly, following the application of acetylcholine and nitroglycerin, both groups showed a significant improvement in all three microvascular perfusion parameters. These results suggest that, despite baseline reductions in both microvascular density and perfusion, human in vivo peripheral microvascular reactivity to both endothelial-dependent and -independent vasoactive agents remains intact in individuals with CAD or multiple risk factors for disease.NEW & NOTEWORTHY To our knowledge, this is the first study to comprehensively characterize in vivo sublingual microvascular structure and function (endothelium-dependent and -independent) in healthy patients and those with CVD. Importantly, we used an easy-to-use handheld device that can be easily translated to clinical settings. Our results indicate that baseline microvascular impairments in structure and function can be detected using the CytoCam technology, although reactivity to acetylcholine may be maintained even during disease in the peripheral microcirculation.

Hyperspectral dark-field microscopy of human breast lumpectomy samples for tumor margin detection in breast-conserving surgery.

Significance: Hyperspectral dark-field microscopy (HSDFM) and data cube analysis algorithms demonstrate successful detection and classification of various tissue types, including carcinoma regions in human post-lumpectomy breast tissues excised during breast-conserving surgeries. Aim: We expand the application of HSDFM to the classification of tissue types and tumor subtypes in pre-histopathology human breast lumpectomy samples. Approach: Breast tissues excised during breast-conserving surgeries were imaged by the HSDFM and analyzed. The performance of the HSDFM is evaluated by comparing the backscattering intensity spectra of polystyrene microbead solutions with the Monte Carlo simulation of the experimental data. For classification algorithms, two analysis approaches, a supervised technique based on the spectral angle mapper (SAM) algorithm and an unsupervised technique based on the K-means algorithm are applied to classify various tissue types including carcinoma subtypes. In the supervised technique, the SAM algorithm with manually extracted endmembers guided by H&E annotations is used as reference spectra, allowing for segmentation maps with classified tissue types including carcinoma subtypes. Results: The manually extracted endmembers of known tissue types and their corresponding threshold spectral correlation angles for classification make a good reference library that validates endmembers computed by the unsupervised K-means algorithm. The unsupervised K-means algorithm, with no a priori information, produces abundance maps with dominant endmembers of various tissue types, including carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma. The two carcinomas' unique endmembers produced by the two methods agree with each other within <2% residual error margin. Conclusions: Our report demonstrates a robust procedure for the validation of an unsupervised algorithm with the essential set of parameters based on the ground truth, histopathological information. We have demonstrated that a trained library of the histopathology-guided endmembers and associated threshold spectral correlation angles computed against well-defined reference data cubes serve such parameters. Two classification algorithms, supervised and unsupervised algorithms, are employed to identify regions with carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma present in the tissues. The two carcinomas' unique endmembers used by the two methods agree to <2% residual error margin. This library of high quality and collected under an environment with no ambient background may be instrumental to develop or validate more advanced unsupervised data cube analysis algorithms, such as effective neural networks for efficient subtype classification.

Ex Vivo Vascular Imaging and Perfusion Studies of Normal Kidney and Tumor Vasculature.

This work describes a comprehensive study of the vascular tree and perfusion characteristics of normal kidney and renal cell carcinoma. Methods: Nephrectomy specimens were perfused ex-vivo, and the regional blood flow was determined by infusion of radioactive microspheres. The vascular architecture was characterized by micronized barium sulphate infusion. Kidneys were subsequently sagitally sectioned, and autoradiograms were obtained to show the perfusate flow in relation to adjacent contact X-ray angiograms. Vascular resistance in defined tissue compartments was quantified, and finally, the tumor vasculature was 3D reconstructed via the micro-CT technique. Results show that the vascular tree of the kidney could be distinctly defined, and autoradiograms disclosed a high cortical flow. The peripheral resistance unit of the whole perfused specimen was 0.78 ± 0.40 (n = 26), while that of the renal cortex was 0.17 ± 0.07 (n = 15 with 114 samples). Micro-CT images from both cortex and medulla defined the vascular architecture. Angiograms from the renal tumors demonstrated a significant vascular heterogeneity within and between different tumors. A dense and irregular capillary network characterized peripheral tumor areas, whereas central parts of the tumors were less vascularized. Despite the dense capillarity, low perfusion through vessels with a diameter below 15 µm was seen on the autoradiograms. We conclude that micronized barium sulphate infusion may be used to demonstrate the vascular architecture in a complex organ. The vascular resistance was low, with little variation in the cortex of the normal kidney. Tumor tissue showed a considerable vascular structural heterogeneity with low perfusion through the peripheral nutritive capillaries and very poor perfusion of the central tumor, indicating intratumoral pressure exceeding the perfusion pressure. The merits and shortcomings of the various techniques used are discussed.

Balancing the Efficiency and Sensitivity of Defect Inspection of Non-Patterned Wafers with TDI-Based Dark-Field Scattering Microscopy.

In semiconductor manufacturing, defect inspection in non-patterned wafer production lines is essential to ensure high-quality integrated circuits. However, in actual production lines, achieving both high efficiency and high sensitivity at the same time is a significant challenge due to their mutual constraints. To achieve a reasonable trade-off between detection efficiency and sensitivity, this paper integrates the time delay integration (TDI) technology into dark-field microscopy. The TDI image sensor is utilized instead of a photomultiplier tube to realize multi-point simultaneous scanning. Experiments illustrate that the increase in the number of TDI stages and reduction in the column fixed pattern noise effectively improve the signal-to-noise ratio of particle defects without sacrificing the detecting efficiency.

Detection of estradiol with a digital immunoassay using an anti-immunocomplex antibody and single-molecule observation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been widely applied to molecular sensors due to their optical properties. We previously reported a molecular detection by observing the scattered light of AuNPs at a single nanoparticle level using dark field microscopy (DFM). Recently, a molecular detection method using digital immunoassay has been reported, taking advantage of the characteristics of DFM. However, the digital immunoassays reported so far have been performed by a conventional sandwich immunoassay, which is difficult to apply to the detection of small molecules. In this study, with the aim of small molecule detection, we developed a digital immunoassay method using an anti-immunocomplex antibody that specifically recognizes immunocomplexes of small molecules with antibodies. The number of AuNPs modified with anti-immunocomplex antibody bound to immunocomplex of estradiol and anti-estradiol antibody was counted at a single nanoparticle level using DFM. We demonstrated for the first time that estradiol molecule can be detected by digital immunoassay using DFM and an anti-immunocomplex antibody with a detection sensitivity of 1 pg/mL.

Gold-Nanorod-Assisted Live Cell Nuclear Imaging Based on Near-Infrared II Dark-Field Microscopy.

Dark-field microscopy offers several advantages, including high image contrast, minimal cell damage, and the absence of photobleaching of nanoprobes, which make it highly advantageous for cell imaging. The NIR-II window has emerged as a prominent research focus in optical imaging in recent years, with its low autofluorescence background in biological samples and high imaging SBR. In this study, we initially compared dark-field imaging results of colorectal cancer cells in both visible and NIR-II wavelengths, confirming the superior performance of NIR-II imaging. Subsequently, we synthesized gold nanorods with localized surface plasmon resonance (LSPR) absorption peaks in the NIR-II window. After bio-compatible modification, we non-specifically labeled colorectal cancer cells for NIR-II dark-field scattering imaging. The imaging results revealed a sixfold increase in SBR, especially in the 1425-1475 nm wavelength range. Finally, we applied this imaging system to perform dark-field imaging of cell nuclei in the NIR-II region and used GNRs for specific nuclear labeling in colorectal cancer cells. The resulting images exhibited higher SBR than non-specifically-labeled cell imaging, and the probe's labeling was precise, confirming the potential application of this system in photothermal therapy and drug delivery for cancer cells.

Plasmonic Nanomaterials in Dark Field Sensing Systems.

Plasma nanoparticles offer promise in data storage, biosensing, optical imaging, photoelectric integration, etc. This review highlights the local surface plasmon resonance (LSPR) excitation mechanism of plasmonic nanoprobes and its critical significance in the control of dark-field sensing, as well as three main sensing strategies based on plasmonic nanomaterial dielectric environment modification, electromagnetic coupling, and charge transfer. This review then describes the component materials of plasmonic nanoprobes based on gold, silver, and other noble metals, as well as their applications. According to this summary, researchers raised the LSPR performance of composite plasmonic nanomaterials by combining noble metals with other metals or oxides and using them in process analysis and quantitative detection.

A Universal Approach to Determine the Atomic Layer Numbers in Two-Dimensional Materials Using Dark-Field Optical Contrast.

Two-dimensional (2D) materials possess unique properties primarily due to the quantum confinement effect, which highly depends on their thicknesses. Identifying the number of atomic layers in these materials is a crucial, yet challenging step. However, the commonly used optical reflection method offers only very low contrast. Here, we develop an approach that shows unprecedented sensitivity by analyzing the brightness of dark-field optical images. The brightness of the 2D material edges has a linear dependence on the number of atomic layers. The findings are modeled by Rayleigh scattering, and the results agree well with the experiments. The relative contrast of single-layer graphene can reach 70% under white-light incident conditions. Furthermore, different 2D materials were successfully tested. By adjusting the exposure conditions, we can identify the number of atomic layers ranging from 1 to over 100. Finally, this approach can be applied to various substrates, even transparent ones, making it highly versatile.

Plasmonic scattering imaging of single Cu2-xSe nanoparticle for Hg2+ detection.

The development of new efficient contrast nanoprobe has been greatly concerned in the field of scattering imaging for sensitive and accurate detection of trace analytes. In this work, the non-stoichiometric Cu2-xSe nanoparticle with typical localized surface plasmon resonance (LSPR) properties originating from their copper deficiency as a plasmonic scattering imaging probe was developed for sensitive and selective detection of Hg2+ under dark-field microscopy. Hg2+ can compete with Cu(I)/Cu(II) which were sources of optically active holes coexisting in these Cu2-xSe nanoparticles for its higher affinity with Se2-. The plasmonic properties of Cu2-xSe were adjusted effectively. Thus, the color scattering images of Cu2-xSe nanoparticles was changed from blue to cyan, and the scattering intensity was obviously enhanced with the dark-field microscopy. There was a linear relationship between the scattering intensity enhancement and the Hg2+ concentration in the range of 10-300 nM with a low detection limit of 1.07 nM. The proposed method has good potential for Hg2+ detection in the actual water samples. This work provides a new perspective on applying new plasmonic imaging probe for the reliable determination of trace heavy metal substances in the environment at a single particle level.

Shaping Copper Oxide Layers on Gold Nanoparticle Ensembles by Controlled Electrodeposition with Single Particle Scatterometry.

Electrodeposition of copper on gold nanoelectrode ensembles result in the formation of uniform copper oxide layers on individual nanoparticles. A linear sweep of voltammetric change induces three distinct morphologies dependent upon particle density. Ex situ imaging and in situ scatterometry at a single-particle level identifies multi-step electrochemical growth sequences that deviated from classical nucleation and growth pathways. In addition, the study demonstrated the possibility of synthesizing sophisticated structures based on the symmetry of nanoelectrodes. This result guides the nanoscale morphology control of electrode ensembles with potential application in electrocatalysis and sensing.

Recent State and Challenges in Spectroelectrochemistry with Its Applications in Microfluidics.

This review paper presents the recent developments in spectroelectrochemical (SEC) technologies. The coupling of spectroscopy and electrochemistry enables SEC to do a detailed and comprehensive study of the electron transfer kinetics and vibrational spectroscopic fingerprint of analytes during electrochemical reactions. Though SEC is a promising technique, the usage of SEC techniques is still limited. Therefore, enough publicity for SEC is required, considering the promising potential in the analysis fields. Unlike previously published review papers primarily focused on the relatively frequently used SEC techniques (ultraviolet-visible SEC and surface-enhanced Raman spectroscopy SEC), the two not-frequently used but promising techniques (nuclear magnetic resonance SEC and dark-field microscopy SEC) have also been studied in detail. This review paper not only focuses on the applications of each SEC method but also details their primary working mechanism. In short, this paper summarizes each SEC technique's working principles, current applications, challenges encountered, and future development directions. In addition, each SEC technique's applicative research directions are detailed and compared in this review work. Furthermore, integrating SEC techniques into microfluidics is becoming a trend in minimized analysis devices. Therefore, the usage of SEC techniques in microfluidics is discussed.

Non-amplification on-spot identifying the sex of dioecious kiwi plants by a portable Raman device.

The kiwi plant is dioecious, and its sex is generally identified from flower morphology at blossoming, which takes several years. It is quite necessary but challenging to on-spot identify the plant sex in juvenile stage. Here the target DNA was obtained by screening the Friendly boy (FrBy) gene which is sex-related for different kiwi plant species. Its complementary sequence was divided into two parts as primer DNA and further attached to different gold nanoparticles (GNPs). The connection between target DNA and primer DNA will promote the formation of plasmonic dimers. Dark field microscopy (DFM) can distinguish particles in different aggregation states. Various conditions were optimized based on the standard of increasing the proportion of dimers while reducing that of large aggregates. Furthermore, two Raman reporters (RR) are separately labeled on the nanoprobes, and the plasmonic dimers lead to a tremendous Raman enhancement of two reporters located at the dimer nanogap. Double-blind tests proved the feasibility of this method on the actual samples of kiwi plant leaves. Our SERS method is sensitive, specific, and reliable for rapid sex identification analysis at the kiwi seeding stage, with great promise for decision-making in field management.

A Novel Relapsing Fever Group Borrelia Isolated from Ornithodoros Ticks of the Brazilian Caatinga.

Tick-borne relapsing fever group (RFG) borreliosis remains neglected as a human disease and little is known on its maintenance in ticks and vertebrates, especially in South America. Therefore, this study investigated borrelial infection in Ornithodoros ticks collected in rodent-inhabited rock formations in the Brazilian semiarid region, within the Caatinga biome. Collected ticks (Ornithodoros rietcorreai and Ornithodoros cf. tabajara) were allowed to feed under laboratory conditions on guinea pigs, which had blood samples examined daily by dark-field microscopy. No spirochetes were visualized in the blood of any of four O. rietcorreai-infested guinea pigs. Contrastingly, spirochetes were visualized between 9 and 39 days after tick feeding in the blood of three guinea pigs, each infested with O. cf. tabajara ticks from a different locality. Guinea pig infection was confirmed by passages into experimental animals and by generating DNA sequences of Borrelia spp. from the blood of spirochetemic guinea pigs. Three O. cf. tabajara populations were infected by the same borrelial organism, which was characterized as a novel RFG agent (named as 'Candidatus Borrelia caatinga') based on 10 Borrelia loci (rrs, flaB, glpQ, gyrB, clpX, pepX, pyrG, recG, rplB and uvrA). We demonstrated that O. cf. tabajara is a competent vector of the novel Borrelia sp. isolates, although none of the infected rodents developed clinical illness.

Multispectral Localized Surface Plasmon Resonance (msLSPR) Reveals and Overcomes Spectral and Sensing Heterogeneities of Single Gold Nanoparticles.

Metal nanoparticles can be sensitive molecular sensors due to enhanced absorption and scattering of light near a localized surface plasmon resonance (LSPR). Variations in both intrinsic properties such as the geometry and extrinsic properties such as the environment can cause heterogeneity in nanoparticle LSPR and impact the overall sensing responses. To date, however, few studies have examined LSPR and sensing heterogeneities, due to technical challenges in obtaining the full LSPR spectra of individual nanoparticles in dynamic assays. Here, we report multispectral LSPR (msLSPR), a wide-field imaging technique for real-time spectral monitoring of light scattering from individual nanoparticles across the whole field of view (FOV) at ∼0.5 nm spectral and ∼100 ms temporal resolutions. Using msLSPR, we studied the spectral and sensing properties of gold nanoparticles commonly used in LSPR assays, including spheres, rods, and bipyramids. Complemented with electron microscopy imaging, msLSPR analysis revealed that all classes of gold nanoparticles exhibited variations in LSPR peak wavelengths that largely paralleled variations in morphology. Compared with the rods and spheres, gold nanobipyramids exhibited both more uniform and stronger sensing responses as long as the bipyramids are structurally intact. Simulations incorporating the experimental LSPR properties demonstrate the negative impact of spectral heterogeneity on the overall performance of conventional, intensity-based LSPR assays and the ability of msLSPR in overcoming both particle heterogeneity and measurement noise. These results highlight the importance of spectral heterogeneity in LSPR-based sensors and the potential advantage of performing LSPR assays in the spectral domain.

Surface Curvature Dominated Guest-Induced Nonequilibrium Deformations of Single Covalent Organic Framework-300 Particles.

Understanding the guest-induced dynamic deformation process of covalent organic frameworks (COFs) is vitally important to further increase their stimulus-response performances. Here we report on the dark-field microscopic (DFM) imaging approach to in situ monitor the guest-induced deformation evolution of individual COF-300 crystals in real time. We observe not only transient and nonequilibrium intermediate deformation states but also local surface curvature-driven diverse adsorption behaviours of single COF-300 particles for dichloromethane (DCM), undergoing one, two, and multiple expansion-contraction deformations as well as contraction-to-expansion transition. The surface curvature-dominated deformations are ascribed to the significant differences in the adsorption capacity for DCM at the curved tip and flat side regions, in which DCM can be adsorbed preferentially by curved tip regions of COF-300.

Probing nanoplastics derived from polypropylene face masks with hyperspectral dark-field microscopy.

The high worldwide consumption of cheap plastic goods has already resulted in a serious environmental plastic pollution, exacerbated by piling of disposed personal protective equipment because of the recent outbreak of COVID-19. The aim of this study was to assess the feasibility of dark-field hyperspectral microscopy in the 400-1000 wavelength range for detection of nanoplastics derived from weathered polypropylene masks. A surgical mask was separated to layers and exposed to UV radiation (254 nm) for 192 h. Oxidative degradation of the polypropylene was evidenced by ATR FT-IR analysis. UV treatment for 192 h resulted in generation of differently shaped micro- and nano-sized particles, visualized by dark-field microscopy. The presence of nanoparticles was confirmed by AFM studies. The hyperspectral profiles (400-1000 nm) were collected after every 48 h of the UV treatment. The distinct hyperspectral features faded after prolonged UV exposure, but the assignment of some particles to either blue or white layers of mask could still be made based on spectral characteristics.

Is There a Role for Dark Field Microscopy in the Diagnosis of Lyme Disease?A Narrative Review.

The diagnosis of Lyme disease is becoming more common in Turkey. Nonetheless, some physicians are not aware of the diagnostic principles that should be followed when faced with a suspected patient and could use tests that are not recommended, such as darkfield microscopy. Dark field microscopy is a diagnostic technique to visualize the spirochetes that cause Lyme disease; however, it is not recommended for the diagnosis of Lyme disease. One of the main limitations of dark field microscopy is its low sensitivity. Another limitation is its high false-positivity rate, as other microorganisms and cellular debris can be mistaken for spirochetes, leading to a misdiagnosis thatmay result in unnecessary treatment. Therefore, this study aimed to review the literature on the role of dark field microscopy as a diagnostic method for Lyme disease and inform physicians about recommended approaches in line with the recommendations of national or international guidelines. An electronic search of Pubmed, Scopus, and Web of Science was performed using the following medical subject headings (MeSH) search terms: Lyme borreliosis, Lyme disease, Borrelia burgdorferi, diagnosis, and microscopy. With this narrative review, we aimed to inform physicians better and improve patient care for patients with suspected Lyme disease.