Single-molecule Detection Sensitivity Research Articles

Abstract 4904: Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology

Abstract Heat induced target retrieval (HITR) is a widely adopted approach for Immunohistochemistry (IHC) and in situ hybridization (ISH) applications on formalin fixed and paraffin embedded (FFPE) tissues to facilitate target access and increase detection sensitivity. A variety of factors, such as fixative type, fixation time, tissue type, epitope abundance, antibody affinity, and probe length greatly affect the efficiency of HITR. Given such variability, over the decades, multiple HITR buffer formulations and protocols were invented to fit the needs in different experimental scenarios. We have developed a novel HITR solution that allows highly efficient target retrieval for RNA ISH application based on the RNAscope technology. When tested on 15 different tumor types, the new HITR solution resulted in significantly better sensitivity and/or tissue morphology comparing to using traditional HITR buffers, including Citrate, EDTA, and Tris based buffers with various pH. Furthermore, this new HITR solution is highly tolerant to sample variability. When used on multiple human tumor tissue microarrays (TMAs), including breast, colon, lung, and gastric cancers, the new HITR solution improved overall signal-to-noise ratio and reduced core disqualification rate, compared to traditional citrate based HITR buffer for ISH. Moreover, when tested on multiple mouse tissues fixed with different types of fixatives for varying periods of time (6 - 72 hours), a single experimental condition with the new HITR solution showed comparable results in both detection sensitivity and morphological integrity in all the samples. It also allowed detection of a target (Tbp) with extremely low abundance (<5 copies/cell). Given that all the RNAscope probes are synthetic oligonucleotide probes with length shorter than 60 bases, the new HITR solution, in combination with the RNAscope technology, will allow universal detection of any RNA targets with single molecule detection sensitivity in FFPE tissues with minimal requirement for analytical condition optimization. Citation Format: Li-chong Wang, Liuliu Pan, Kuang-Jung Chang, Daniel Kim, Xingyong Wu, Hongwei Wang, Casey Kernag, Bingqing Zhang, Mingxiao He, Nan Su, Xiao-Jun Ma, Yuling Luo. Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4904. doi:10.1158/1538-7445.AM2015-4904

All-nanophotonic NEMS biosensor on a chip.

Integrated chemical and biological sensors give advantages in cost, size and weight reduction and open new prospects for parallel monitoring and analysis. Biosensors based on nanoelectromechanical systems (NEMS) are the most attractive candidates for the integrated platform. However, actuation and transduction techniques (e.g. electrostatic, magnetomotive, thermal or piezoelectric) limit their operation to laboratory conditions. All-optical approach gives the possibility to overcome this problem, nevertheless, the existing schemes are either fundamentally macroscopic or excessively complicated and expensive in mass production. Here we propose a novel scheme of extremely compact NEMS biosensor monolithically integrated on a chip with all-nanophotonic transduction and actuation. It consists of the nanophotonic waveguide and the nanobeam cantilever placed above the waveguide, both fabricated in the same CMOS-compatible process. Being in the near field of the strongly confined photonic or plasmonic mode, cantilever is efficiently actuated and its response is directly read out using the same waveguide, which results in a very high sensitivity and capability of single-molecule detection even in atmosphere.

Coherent anti-Stokes Raman scattering with single-molecule sensitivity using a plasmonic Fano resonance

Plasmonic nanostructures are of particular interest as substrates for the spectroscopic detection and identification of individual molecules. Single-molecule sensitivity Raman detection has been achieved by combining resonant molecular excitation with large electromagnetic field enhancements experienced by a molecule associated with an interparticle junction. Detection of molecules with extremely small Raman cross-sections (~10(-30) cm(2) sr(-1)), however, has remained elusive. Here we show that coherent anti-Stokes Raman spectroscopy (CARS), a nonlinear spectroscopy of great utility and potential for molecular sensing, can be used to obtain single-molecule detection sensitivity, by exploiting the unique light harvesting properties of plasmonic Fano resonances. The CARS signal is enhanced by ~11 orders of magnitude relative to spontaneous Raman scattering, enabling the detection of single molecules, which is verified using a statistically rigorous bi-analyte method. This approach combines unprecedented single-molecule spectral sensitivity with plasmonic substrates that can be fabricated using top-down lithographic strategies.

Split-FP Conjugated Metal Nanoparticle Raman Nanoprobes for Ultra-Sensitive Molecular Detection

The recent convergence of biotechnology and nanotechnology now provides unique means to develop hybrid nanomaterials for advanced biosensing applications. In particular, the controlled assembly of metal gold (Au) and silver (Ag) nanoparticles using bio-inspired scaffolds is a promising approach for the design of novel Surface Enhanced Raman Spectroscopy (SERS) nanoprobes capable of single molecule detection sensitivity. Here, we propose a new design for colloidal Raman nanoprobes based on split-FP/metal nanoparticle hybrid composites. We use split fluorescence proteins (split-FPs) as both activatable Raman reporters and molecular glue to assemble Au or Ag nanoparticles into photonically-active SERS nanoclusters. We expressed recombinant split-FPs (sGFP, sYFP, sCFP) with metal binding domains and engineered their complementary peptide fragments for oriented and controlled grafting at the surface of Au and Ag nanoparticles. These split-FPs fragments were respectively coated on various size of metal nanoparticles (5-50nm diameter), and were characterized by electrophoresis, HPLC, Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), and immunoblotting. In vitro, complementary metal nanoparticles functionalized with split-FP fragments can self-assemble and form nanoclusters as confirmed by electrophoresis and TEM measurements. To improve the complementation kinetics of split-FP/peptide pairs on nanoparticles, various point mutations were introduced in split-FPs. We further show that SERS signals from split-FPs are specifically detected upon biomolecular interactions of split-FP fragments and that the unique Raman signature of the FP chromophores is easily distinguished over other protein modes when bound to plasmonic nanomaterials. These results demonstrate the potential for highly selective and sensitive SERS detection based on split-FP/metal nanoparticle hybrid probes.

Super-Resolution Imaging in Plant Cells

In the past few years, the emergence of super-resolution microscopy techniques enabled diffraction unlimited imaging using the same diffraction-limited far-field optics as in conventional fluorescence microscopy. One approach, implemented in stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM) and fluorescence photoactivation microscopy (FPALM), relies on the stochastic nature of single-molecule switching. High spatial resolution in these techniques can only be realized when single fluorophores are detected with sufficiently high signal to noise ratios to allow localization with nanometer accuracy. Though researchers have started new journey of understanding the biological process in mammalian cells, yet very few reports exist of studying biological process in plant cells or tissues with such high spatial resolution. The primary reasons are the high autofluorescence of endogenous components and the presence of thick cell walls. We have conducted careful assessment of existing imaging techniques for single molecule imaging in plant cells and tissues, which is the fundamental aspect of super-resolution imaging. To improve single-molecule detection sensitivity, we are developing new microscopy techniques based on the concept of light sheet illumination. Using these new microscopes, we are trying to resolve nanoscale cellular structures in plant seedlings.

Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples

Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification.

Optimizing Photophysical Conditions in Confocal Fluorescence Correlation Spectroscopy (FCS)

FCS is a powerful method to measure diffusional and rotational properties of molecules as well as intramolecular transitions (e.g. triplet transition rates and isomerization) and intermolecular interactions (e.g. protein-protein, DNA-protein, etc.). Since the introduction of the confocal measuring principle by Rigler et al. and single photon counting avalanche photodiodes, FCS techniques attained a tremendous revival due to an increased sensitivity enabling even the study of single fluorescent molecules traversing the open, optically confined volume element. Despite these improvements it still remains desirable to further enhance the most important parameter in FCS measurements, i.e. the count rate per molecule (CRpM), in order to further improve the overall performance of FCS measurements (e.g. to enhance signal-to-noise and signal-to-background ratios, and, simultaneously, to minimize acquisition times).To this end we present here investigations showing an increase of the CRpM of > 200 kHz for Rhodamine Green (excit. laser power > 0.4 mW, 488 nm) along with an effective confocal volume in the subfemtoliter range (0.76 fl). However, photophysical processes like photobleaching, saturation, Rayleigh and Raman scattering as well as triplet transitions and/or isomerization have to be taken into account to perform optimized FCS measurements. Under these conditions we succeeded to yield signal-to-background ratios (i.e. CRpM/BGCR with BGCR background count rate due to scatter) of > 400 at relatively low laser power (0.1-0.3 mW). Further increasing the laser power results in increased triplet transition rates, bleaching and linearly increasing BGCR whereas CRpM levels out into a plateau. Thus, higher excitation energies (above 0.5mW) are in case of Rhodamine Green of counterproductivity. Finally, thanks to the improved single molecule detection sensitivity, we demonstrate that pico- and even femtomolar concentrations of fluorophores can be detected by confocal FCS under optimized photophysical conditions.

Field programmable gate array based reconfigurable scanning probe/optical microscope

The increasing popularity of nanometrology and nanospectroscopy has pushed researchers to develop complex new analytical systems. This paper describes the development of a platform on which to build a microscopy tool that will allow for flexibility of customization to suit research needs. The novelty of the described system lies in its versatility of capabilities. So far, one version of this microscope has allowed for successful near-field and far-field fluorescence imaging with single molecule detection sensitivity. This system is easily adapted for reflection, polarization (Kerr magneto-optical (MO)), Raman, super-resolution techniques, and other novel scanning probe imaging and spectroscopic designs. While collecting a variety of forms of optical images, the system can simultaneously monitor topographic information of a sample with an integrated tuning fork based shear force system. The instrument has the ability to image at room temperature and atmospheric pressure or under liquid. The core of the design is a field programmable gate array (FPGA) data acquisition card and a single, low cost computer to control the microscope with analog control circuitry using off-the-shelf available components. A detailed description of electronics, mechanical requirements, and software algorithms as well as examples of some different forms of the microscope developed so far are discussed.

Individual gold dimers investigated by far‐ and near‐field imaging

Plasmon resonances in 3D nanoparticle arrangements can produce strong localized optical fields, which are of importance for any application involving interaction of light with subwavelength volumes of matter down to the molecular level. In particular, remarkable field enhancement and confinement occur in a dimer geometry formed by two identical closely spaced particles. Although, recent advances in nanofabrication have rendered the fabrication of complex plasmon architectures more accessible, addressing their local fields in a nonperturbative fashion remains not straightforward, because metallic nanostructures are rather sensitive to their local environment. Here we study gold dimers fabricated by e-beam lithography. Individual dimers are imaged both by far- and near-field methods. First, the near-field electromagnetic interaction in an ensemble of dimers is investigated by scattering spectroscopy, using dark field microscopy. Next, to probe their local field, we explore the luminescence of individual gold dimers utilizing a confocal microscope with single molecule detection sensitivity. We provide a statistical analysis of the dimer luminescence for different incident polarizations, with direct comparison to single particles (monomers). Finally, the near-field transmission of the resonant dimers is mapped with a subwavelength resolution using polarized controlled near-field scanning optical microscopy. Surprisingly, no clear evidence of the high mode density in the dimer gap is observed. This result may be attributed to the limited coupling of the field emitted by the aperture probe to the dimer mode.

Cover Picture: Nanoscale Organization of the Pathogen Receptor DC‐SIGN Mapped by Single‐Molecule High‐Resolution Fluorescence Microscopy (ChemPhysChem 10/2007)

The cover picture shows a high-resolution fluorescence image (12×7 μm2) of the pathogen recognition receptor DC-SIGN expressed on the membrane of an immature dendritic cell. Simultaneously the topography (gray) and the fluorescence (color) image have been obtained using near-field scanning optical microscopy (NSOM; lower left panel). Because spatial resolution and single-molecule detection sensitivity are increased, NSOM is capable to resolve individual domains of DC-SIGN of typically 185 nm in size that are randomly distributed over the whole cell membrane (lower right panel). Find out more in the Article by de Bakker et al. on page 1473.

Single-molecule detection sensitivity using planar integrated optics on a chip

We present a fully planar integrated optical approach to single-molecule detection based on microfabricated planar networks of intersecting solid and liquid-core waveguides. We study fluorescence from dye molecules in liquid-core antiresonant reflecting optical waveguides, and demonstrate subpicoliter excitation volumes, parallel excitation through multiple pump waveguides, and single-molecule detection sensitivity. Integrated silicon photonics combined with single-molecule detection in solution create a compact, robust, and sensitive platform that has applications in numerous fields ranging from atomic physics to the life sciences.

Cross Talk Free Fluorescence Cross Correlation Spectroscopy in Live Cells

Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin.

Near-Field Fluorescence Microscopy An Optical Nanotool to Study Protein Organization at the Cell Membrane

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail and accuracy.

Near-Field Fluorescence Microscopy: An Optical Nanotool to Study Protein Organization at the Cell Membrane

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail and accuracy.

Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dendritic cells

Clustering of cell surface receptors into micro-domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near-field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C-type lectin DC-SIGN on the membrane of dendritic cells, both in air and in liquid.

Ligand-receptor interactions in live cells by fluorescence correlation spectroscopy.

Receptor binding studies most often require the use of radioactively labeled ligands. In certain cases, the numbers of receptors are few per cell and no specific binding is detected because of a high background. Specific interactions between certain ligands (e.g. peptides, hormones, natural products) and their receptors are, therefore, often overlooked by the conventional binding technique. Fluorescence correlation spectroscopy (FCS) allows detection of the interaction of ligands with receptors in their native environment in live cells in a tiny confocal volume element (0.2 fl) at single-molecule detection sensitivity. This technique permits the identification of receptors which were not possible before to detect by isotope labeling. The beauty of the FCS technique is that there is no need for separating an unbound ligand from a bound one to calculate the receptor bound and free ligand fractions. This review will show FCS as a sensitive and a rapid technique to study ligand-receptor interaction in live cells and will demonstrate that the FCS-analysis of ligand-receptor interactions in live cells fulfils all the criteria of a ligand binding to its receptor i.e. it is able to provide information on the affinity and specificity of a ligand, binding constant, association and dissociation rate constants as well as the number and mobility of receptors carrying a fluorescently labeled ligand. This review is of pharmaceutical significance since it will provide insights on how FCS can be used as a rapid technique for studying ligand-receptor interactions in cell cultures, which is one step forward towards a high throughput drug screening in cell cultures.

Ultrasensitive coincidence fluorescence detection of single DNA molecules.

We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.

Cell biology beyond the diffraction limit: near-field scanning optical microscopy.

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution--approximately 250 nm--dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future.

Insulin binding monitored by fluorescence correlation spectroscopy.

The characteristics of insulin binding to its receptors have been extensively studied by the radioligand binding assay. We used fluorescence correlation spectroscopy to determine the distribution of diffusion times and further novel data on the kinetics of insulin's binding to its receptor. Cultured human renal tubular cells (HRTC) were incubated with tetramethyl rhodamine labelled insulin (Rh-Ins) for 60 min. Fluorescence intensity fluctuations and autocorrelation functions for Rh-Ins, free in the incubation medium and bound to the cell membrane, were studied at single-molecule detection sensitivity in a 0.2 fL confocal volume. Measurements at the cell membrane revealed Rh-Ins binding with at least two diffusion components (diffusion times tauD1 = 0.8 ms, tauD2 = 20 ms) and corresponding weight fractions of y1 = 0.43 and y2 = 0.42. Specificity of the binding was shown by the dislocation of bound Rh-Ins when excess unlabelled insulin was added. Scatchard analysis showed a nonlinear plot, revealing two binding processes with different affinities (Kass approximately 2 x 10(10) M(-1) and approximately 1 x 10(9) M(-1), respectively). The fluorescence correlation spectroscopy results show two classes of binding sites with different affinities for insulin, or interactions between receptor sites consistent with negative cooperativity. This conclusion is in agreement with studies of insulin binding using radioligand binding assays. Because of its high sensitivity (single molecule detection), FCS, provides additional data allowing a more precise evaluation of the kinetics of ligand-receptor interactions at low expression levels in living cells.