ABSTRACT
Observing the hybridisation kinetics of DNA probes immobilised on plasmonic nanoparticles is key in plasmon-enhanced fluorescence detection of weak emitting species, and refractive index based single-molecule detection on optoplasmonic sensors. The role of the local field in providing plasmonic signal enhancements for single-molecule detection has been studied in great detail. Nevertheless, few studies have compared the experimental results in both techniques for single-molecule studies. Here we developed the first optical setup that integrates optoplasmonic and DNA-PAINT based detection of oligonucleotides to compare these sub-platforms and provide complementary insights into single molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.
Subject(s)
Gold , Nanotubes , Kinetics , Gold/chemistry , Single Molecule Imaging , Surface Plasmon Resonance/methods , Nanotubes/chemistry , DNA/genetics , DNA/chemistryABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100's of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.
ABSTRACT
May-Grünwald-Giemsa (MGG) stain is a Romanowsky-type, polychromatic stain as those of Giemsa, Leishman and Wright. Apart being the reference method of haematology, it has become a routine stain of diagnostic cytopathology for the study of air-dried preparations (lymph node imprints, centrifuged body fluids and fine needle aspirations). In the context of their actions of promoting the principles of quality assurance in cytopathology, the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP) and the French Society of Clinical Cytology (SFCC) conducted a proficiency test on MGG stain in 2013. Results from the test, together with the review of literature data allow pre-analytical and analytical steps of MGG stain to be updated. Recommendations include rapid air-drying of cell preparations/imprints, fixation using either methanol or May-Grünwald alone for 3-10minutes, two-step staining: 50% May-Grünwald in buffer pH 6.8 v/v for 3-5minutes, followed by 10% buffered Giemsa solution for 10-30minutes, and running water for 1-3minutes. Quality evaluation must be performed on red blood cells (RBCs) and leukocytes, not on tumour cells. Under correct pH conditions, RBCs must appear pink-orange (acidophilic) or buff-coloured, neither green nor blue. Leukocyte cytoplasm must be almost transparent, with clearly delineated granules. However, staining may vary somewhat and testing is recommended for automated methods (slide stainers) which remain the standard for reproducibility. Though MGG stain remains the reference stain, Diff-Quik(®) stain can be used for the rapid evaluation of cell samples.
Subject(s)
Coloring Agents , Cytodiagnosis/standards , Eosine Yellowish-(YS) , Methylene Blue , Practice Guidelines as Topic , Staining and Labeling/methods , Automation , Azure Stains , Cell Biology/organization & administration , Coloring Agents/chemistry , Cytodiagnosis/methods , Eosine Yellowish-(YS)/chemistry , Erythrocytes/ultrastructure , France , Humans , Hydrogen-Ion Concentration , Leukocytes/ultrastructure , Methylene Blue/chemistry , Organelles/ultrastructure , Quality Assurance, Health Care , Reproducibility of Results , Societies, Scientific , Staining and Labeling/instrumentation , Staining and Labeling/standards , Tissue Fixation/methods , XanthenesABSTRACT
We outline a full-vectorial three-dimensional multi-mode matching technique in a cylindrical coordinate system that addresses the mutual coupling among multiple modes co-propagating in a perturbed whispering gallery mode microcavity. In addition to its superior accuracy in respect to our previously implemented single-mode matching technique, this current technique is suitable for modelling waveguide-to-cavity coupling where the influence of multi-mode coupling is non-negligible. Using this methodology, a robust scheme for hybrid integration of a microcavity onto a silicon-on-insulator platform is proposed.
ABSTRACT
A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing its free spectral range (FSR), the module is put in parallel with a fiber taper-microcavity system and then calibrated through isolating and eliminating random shifts in the laser frequency (i.e. laser jitter noise). To realize the taper-microcavity junction and to maximize the optical power that is transferred to the resonator, a single-mode optical fiber waveguide is pulled. Solutions containing polystyrene nanobeads are then prepared and flown to the microcavity in order to demonstrate the system's ability to sense binding to the surface of the microcavity. Data is post-processed via adaptive curve fitting, which allows for high-resolution measurements of the quality factor as well as the plotting of time-dependent parameters, such as resonant wavelength and split frequency shifts. By carefully inspecting steps in the time-domain response and shifting in the frequency-domain response, this instrument can quantify discrete binding events.
Subject(s)
Interferometry/instrumentation , Interferometry/methods , Nanotechnology/instrumentation , Nanotechnology/methods , Electronic Data Processing , Reference ValuesABSTRACT
We present a full-vectorial three-dimensional whispering-gallery-mode microcavity analysis technique. With this technique, optical properties such as resonance wavelength, quality factor, and electromagnetic field distribution of a microcavity in the presence of individual nanoparticle adsorption can be simulated with high accuracy, even in the presence of field distortion from plasmon effects at a wavelength close to plasmon resonance. This formulation is applicable to a wide variety of whispering-gallery related problems, such as waveguide to cavity coupling and full wave propagation analysis of a general whispering-gallery-mode microcavity where axisymmetry along the azimuthal direction is not required.
ABSTRACT
We simulate light propagation in perturbed whispering-gallery mode microcavities using a two-dimensional finite-difference beam propagation method in a cylindrical coordinate system. Optical properties of whispering-gallery microcavities perturbed by polystyrene nanobeads are investigated through this formulation. The light perturbation as well as quality factor degradation arising from cavity ellipticity are also studied.
ABSTRACT
The 2001 Bethesda System is a uniform system of terminology for reporting results of pap smears. It is acknowledged by most cytopathologists worldwide as a standard for cervical cytology reports. In France, several national surveys have confirmed its current utilization. However, more specific analysis have shown that the Bethesda System may be routinely modified by individual laboratories or even individual cytopathologist working within the same department. The aim of this progress report was to emphasize the importance of fully understanding the Bethesda System and applying it in a rigorous and standardized way.
