Ultrasensitive detection and characterization of single nanoparticles (<100 nm) is important in nanotechnology and life sciences. Direct measurement of the elastically scattered light from individual nanoparticles represents the simplest and the most direct method for particle detection. However, the sixth-power dependence of scattering intensity on particle size renders very small particles indistinguishable from the background. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, here we report the development of high sensitivity flow cytometry (HSFCM) that achieves real-time light-scattering detection of single silica and gold nanoparticles as small as 24 and 7 nm in diameter, respectively. This unprecedented sensitivity enables high-resolution sizing of single nanoparticles directly based on their scattered intensity. With a resolution comparable to that of TEM and the ease and speed of flow cytometric analysis, HSFCM is particularly suitable for nanoparticle size distribution analysis of polydisperse/heterogeneous/mixed samples. Through concurrent fluorescence detection, simultaneous insights into the size and payload variations of engineered nanoparticles are demonstrated with two forms of clinical nanomedicine. By offering quantitative multiparameter analysis of single nanoparticles in liquid suspensions at a throughput of up to 10?000 particles per minute, HSFCM represents a major advance both in light-scattering detection technology and in nanoparticle characterization.
An electrochemical approach to the intramolecular aminooxygenation of unactivated alkenes has been developed. This process is based on the addition of nitrogen-centered radicals, generated through electrochemical oxidation, to alkenes followed by trapping of the cyclized radical intermediate with 2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPO). Difunctionalization of a variety of alkenes with easily available carbamates/amides and TEMPO affords aminooxygenation products in high yields and with excellent trans selectivity for cyclic systems (d.r. up to>20:1). The approach provides a much-needed complementary route to existing cis-selective methods.
Mitochondria play a pivotal role in determining the point-of-no-return of the apoptotic process. Therefore, anticancer drugs that directly target mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. In this study, we report the development of an in vitro strategy to quickly identify the therapeutic agents that induce apoptosis via directly affecting mitochondria. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (??(m)) fluorescence of individual mitochondria using a laboratory-built high-sensitivity flow cytometer. The feasibility of this method was tested with eight widely used anticarcinogens. Dose-dependent ??(m) losses were observed for paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, and triptolide, but not for cisplatin or actinomycin D, which agrees well with their mechanisms of apoptosis induction reported in the literature. The as-developed method offers an effective approach to identify mitochondria-targeting anticancer compounds.
A scanning angle (SA) Raman microscope with 532-nm excitation is reported for probing chemical content perpendicular to a sample interface. The instrument is fully automated to collect Raman spectra across a range of incident angles from 20.50 to 79.50° with an angular spread of 0.4±0.2° and an angular uncertainty of 0.09°. Instrumental controls drive a rotational stage with a fixed axis of rotation relative to a prism-based sample interface mounted on an inverted microscope stage. Three benefits of SA Raman microscopy using visible wavelengths, compared to near infrared wavelengths are: (i) better surface sensitivity; (ii) increased signal due to the frequency to the fourth power dependence of the Raman signal, and the possibility for resonant enhancement; (iii) the need to scan a reduced angular range to shorten data collection times. These benefits were demonstrated with SA Raman measurements of thin polymer films of polystyrene or a diblock copolymer of polystyrene and poly(3-hexylthiophene-2,5-diyl). Thin film spectra were collected with a signal-to-noise ratio of 30 using a 0.25 s acquisition time.
Advanced methods are urgently needed to determine the identity and viability of trace amounts of pathogenic bacteria in a short time. Existing approaches either fall short in the accurate assessment of microbial viability or lack specificity in bacterial identification. Bacteriophages (or phages for short) are viruses that exclusively infect bacterial host cells with high specificity. As phages infect and replicate only in living bacterial hosts, here we exploit the strategy of using tetracysteine (TC)-tagged phage in combination with biarsenical dye to the discriminative detection of viable target bacteria from dead target cells and other viable but nontarget bacterial cells. Using recombinant M13KE-TC phage and Escherichia coli ER2738 as a model system, distinct differentiation between individual viable target cells from dead target cells was demonstrated by flow cytometry and fluorescence microscopy. As few as 1% viable E. coli ER2738 can be accurately quantified in a mix with dead E. coli ER2738 by flow cytometry. With fluorescence microscopic measurement, specific detection of as rare as 1 cfu/mL original viable target bacteria was achieved in the presence of a large excess of dead target cells and other viable but nontarget bacterial cells in 40 mL artificially contaminated drinking water sample in less than 3 h. This TC-phage-FlAsH approach is sensitive, specific, rapid, and simple, and thus shows great potential in water safety monitoring, health surveillance, and clinical diagnosis of which trace detection and identification of viable bacterial pathogens is highly demanded.
The single particle orientation and rotational tracking (SPORT) techniques have seen rapid development in the past 5 years. Recent technical advances have greatly expanded the applicability of SPORT in biophysical studies. In this feature article, we survey the current development of SPORT and discuss its potential applications in biophysics, including cellular membrane processes and intracellular transport.
Single-cell analysis is vital in providing insights into the heterogeneity in molecular content and phenotypic characteristics of complex or clonal cell populations. As many essential proteins and most transcription factors are produced at a low copy number, analytical tools with superior sensitivity to enable the analysis of low abundance proteins in single cells are in high demand. ?-galactosidase (?-gal) has been the standard cellular reporter for gene expression in both prokaryotic and eukaryotic cells. Here we report the development of a high-throughput method for the single-cell analysis of low copy number ?-gal proteins using a laboratory-built high-sensitivity flow cytometer (HSFCM). Upon fluorescence staining with a fluorogenic substrate, quantitative measurements of the basal and near-basal expression of ?-gal in single Escherichia coli BL21(DE3) cells were demonstrated. Statistical distribution can be determined quickly by analyzing thousands of individual cells in 1-2min, which reveals the heterogeneous expression pattern that is otherwise masked by the ensemble analysis. Combined with the quantitative fluorometric assay and the rapid bacterial enumeration by HSFCM, the ?-gal expression distribution profile could be converted from arbitrary fluorescence units to protein copy numbers per cell. The sensitivity and speed of the HSFCM offers great capability in quantitative analysis of low abundance proteins in single cells, which would help gaining a deeper insight into the heterogeneity and fundamental biological processes in microbial populations.
Employing single nanoparticle detection with a laboratory-built high-sensitivity flow cytometer, we developed a simple and versatile platform that is capable of detecting the surface plasmon resonance scattering of gold nanoparticles (GNPs) as small as 24 nm, differentiating GNPs of different sizes, and providing accurate quantification of GNPs. Low-concentration samples (fM to pM) in small volumes (microL) can be measured in minutes with an analysis rate of up to 100-200 GNPs per second. Among these features, absolute quantification provides a distinct advantage because it does not require standard samples.
A compact, high-sensitivity, dual-channel flow cytometer (HSDCFCM) was developed for the individual analysis of nanosized particles and biomolecules. A hydrodynamic focusing technique was applied to confine the sample stream and enable small probe volume. Fluorescence bursts from single R-phycoerythrin (R-PE) molecules passing through the laser beam were well resolved from the background with signal-to-noise ratio of 17. Excellent size discrimination was demonstrated with a mixture of three sizes of polystyrene nanoparticles. Simultaneous measurement of fluorescence and light scattering signals from individual nanoparticles was demonstrated with the 100 nm fluorescent latex beads. Doxorubicin-loaded ZrO(2) nanoparticles and fluorescently stained Escherichia coli ER2738 cells were analyzed successfully with dual-channel detection. Particle counting is demonstrated with the 210 nm fluorescent latex beads, and excellent correlation (R(2) > 0.998) between the manufacturer-reported concentrations and those measured by HSDCFCM enumeration was obtained. The measured sample detection efficiency was approximately 90% on average for particle concentrations ranging from 1.62 x 10(5) to 3.93 x 10(7) particles/mL. Sample mixtures with varying proportions of fluorescently labeled and unlabeled nanoparticles were also analyzed with good ratio correspondence. By providing rapid, quantitative, and multiparameter characterization of nanoparticles, it is believed that the HSDCFCM will find many applications in the fields of bionanotechnology, bioanalytical chemistry, and biomedicine.
Mitochondria are one of the most important organelles responsible for cellular energy metabolism and apoptosis regulation. However, single-mitochondrion analysis is challenging, because of their small sizes and the low content of organelle constituents. Here, we report the development of a sensitive and versatile platform for high-throughput multiparameter analysis of individual mitochondria. Employing specific fluorescent staining with a laboratory-built high-sensitivity flow cytometer (HSFCM), we demonstrate the simultaneous detection of side scatter, cardiolipin, and mitochondria DNA (mtDNA) of a single mitochondrion. Simultaneous measurements of side scatter, porin, and cytochrome c of individual mitochondria are reported for the first time. Correlation analysis among multiple attributes on an organelle-by-organelle basis could provide a more definitive assessment of the purity, structure integrity, and apoptosis-related proteins of isolated mitochondria than bulk measurement. This work represents a significant advancement in single-mitochondrion analysis. We believe that the HSFCM holds great potential for studying apoptotic signal transduction pathways at the single-mitochondrion level.
Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.
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