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ABSTRACT: In stimulated emission depletion (STED)-based or up-conversion depletion-based super-resolution optical microscopy, the donut-shaped depletion beam profile is of critical importance to its resolution. In this study, we investigate the transformation of the donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective, and model STED point spread function (PSF) as a function of donut beam profile. We show experimentally that the intensity profile of the dark kernel of the donut can be approximated as a parabolic function, whose slope is determined by the donut beam size before the objective back aperture, or the effective NA. Based on this, we derive the mathematical expression for continuous wave (CW) STED PSF as a function of focal plane donut and excitation beam profiles, as well as dye properties. We find that the effective NA and the residual intensity at the center are critical factors for STED imaging quality and the resolution. The effective NA is critical for STED resolution in that it not only determines the donut shape but also the area the depletion laser power is dispersed. An improperly expanded depletion beam will have negligible improvement in resolution. The polarization of the depletion beam also plays an important role as it affects the residual intensity in the center of the donut. Finally, we construct a CW STED microscope operating at 488 nm excitation and 592 nm depletion with a resolution of 70 nm. Our study provides detailed insight to the property of donut beam, and parameters that are important for the optimal performance of STED microscopes. This paper will provide a useful guide for the construction and future development of STED microscopes.
The Review of scientific instruments 04/2013; 84(4):043701. · 1.52 Impact Factor
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Anthony S Stender,
Kyle Marchuk,
Chang Liu,
Suzanne Sander,
Matthew W Meyer,
Emily A Smith,
Bhanu Neupane, Gufeng Wang,
Junjie Li,
Ji-Xin Cheng,
Bo Huang,
Ning Fang
Chemical Reviews 02/2013; · 40.20 Impact Factor
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ABSTRACT: Super-localization of single molecules and nanoparticles has become an essential procedure to bring new insights into nanoscale structures and dynamics of biological systems. In the present study, super-localization is combined with the newly introduced differential interference contrast (DIC) microscopy-based single particle orientation and rotational tracking (SPORT). The new technique overcomes the difficulty in localization of the anti-symmetric DIC point spread function by using a dual-modality microscope configuration for simultaneous rotational tracking and localization of single gold nanorods with nanometer-scale precision. The new imaging setup has been applied to study the steric hin-drance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment. This technique has great potential in the study of biological processes where both localization and rotational information are required.
ACS Nano 01/2013; · 10.77 Impact Factor
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ABSTRACT: A full understanding of cell mechanics requires knowledge of both translational and rotational dynamics. The single particle orientation and rotational tracking (SPORT) technique is combined here with correlation analysis to identify the fundamental rotational modes: in-plane rotation and out-of-plane tilting, as well as other more complex rotational patterns, from the vast image data captured at a temporal resolution of 5 ms for single gold nanorod probes in live cell imaging experiments. The unique capabilities of visualizing and understanding rotational motions of functional nanoparticles on live cell membranes allow correlation of the rotational and translational dynamics in unprecedented detail and provide new insights into complex membrane processes. Particles with functionalized surfaces, which interact with the membrane in fundamentally different ways, can exhibit distinct rotational modes and are, for the first time, directly visualized, and these show the early events for membrane approach and attachment.
Small 11/2012; · 8.35 Impact Factor
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ABSTRACT: Direct visualization of axonal transport in live neurons is essential for our understanding of the neuronal functions and the working mechanisms of microtubule-based motor proteins. Here we use the high-speed single particle orientation and rotational tracking technique to directly visualize the rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport, with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause.
Nature Communications 08/2012; 3:1030. · 7.40 Impact Factor
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ABSTRACT: Keeping track: By combining differential interference contrast (DIC) image pattern recognition with DIC polarization anisotropy, the exact full three-dimensional angular information of individual tilted gold nanorods positioned in the focal plane of the objective lens can be readily determined. The angular rotational modes and kinetics of individual in-focus gold nanorods can thus be resolved dynamically.
Angewandte Chemie International Edition 06/2012; 51(31):7734-8. · 13.45 Impact Factor
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ABSTRACT: Researchers rely on a variety of microscopic techniques for observing and tracking anisotropic nanoparticles in real time experiments. This technical note focuses on the optical behavior exhibited by gold nanorods at nonplasmonic wavelengths under differential interference contrast microscopy (DIC). Intense diffraction patterns appear at nonplasmonic wavelengths, and the behavior of these patterns can be altered by adjusting the surrounding medium or the polarizer setting. Such patterns are absent when linear and crossed polarizations are utilized. Making polarization adjustments is important in DIC microscopy, because it affects bias retardation and image contrast. The nonplasmonic diffraction bands that were observed could potentially be exploited for rotational tracking, but more importantly, researchers should exhibit care in selecting a nanorod sample and the polarization setting when working with DIC microscopy.
Analytical Chemistry 05/2012; 84(12):5210-5. · 5.86 Impact Factor
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ABSTRACT: We introduce a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. We demonstrated the localization precision of 4-7 nm laterally and 16 nm axially for 40-nm gold nanospheres at an imaging rate of 10 frames per second. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events.
Analytical Chemistry 04/2012; 84(9):4111-7. · 5.86 Impact Factor
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ABSTRACT: Precisely imaging and tracking dynamic biological processes in live cells are crucial for both fundamental research in life sciences and biomedical applications. Nonfluorescent nanoparticles are emerging as important optical probes in live-cell imaging because of their excellent photostability, large optical cross sections, and low cytotoxicity. Here, we provide a review of recent development in optical imaging of nonfluorescent nanoparticle probes and their applications in dynamic tracking and biosensing in live cells. A brief discussion on cytotoxicity of nanoparticle probes is also provided.
Methods in enzymology 01/2012; 504:83-108. · 1.90 Impact Factor
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ABSTRACT: The single particle orientation and rotational tracking (SPORT) technique was introduced recently to follow the rotational motion of plasmonic gold nanorod under a differential interference contrast (DIC) microscope. In biological studies, however, cellular activities usually involve a multiplicity of molecules; thus, tracking the motion of a single molecule/object is insufficient. Fluorescence-based techniques have long been used to follow the spatial and temporal distributions of biomolecules of interest thanks to the availability of multiplexing fluorescent probes. To know the type and number of molecules and the timing of their involvement in a biological process under investigation by SPORT, we constructed a dual-modality DIC/fluorescence microscope to simultaneously image fluorescently tagged biomolecules and plasmonic nanoprobes in living cells. With the dual-modality SPORT technique, the microtubule-based intracellular transport can be unambiguously identified while the dynamic orientation of nanometer-sized cargos can be monitored at video rate. Furthermore, the active transport on the microtubule can be easily separated from the diffusion before the nanocargo docks on the microtubule or after it undocks from the microtubule. The potential of dual-modality SPORT is demonstrated for shedding new light on unresolved questions in intracellular transport.
Analytical Chemistry 12/2011; 84(2):1134-8. · 5.86 Impact Factor
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ABSTRACT: An automatic calibration and angle-scanning prism-type total internal reflection fluorescence microscope (TIRFM) was modified to function in both TIRFM and pseudo-TIRFM modes. When the incident angle of the excitation laser beam was controlled to be larger than the critical angle, the instrument served as a variable-angle TIRFM. A homemade computer program automatically calibrates the laser illumination spot in the sample to overlap with the center of the microscope's field of view. Then, by measuring the fluorescence intensities at different incident angles, the z-positions of fluorescent nanospheres close to the cell basolateral membrane can be extracted. When the incident angle is reduced to be in the subcritical range, the instrument works as a pseudo-TIRFM. The whole cell body from bottom to top can be imaged in a vertical scan process. Furthermore, the illumination field depth in the pseudo-TIRFM can be controlled by changing the incident angle or the horizontal position of the laser spot.
Journal of the Association for Laboratory Automation 08/2011; 16(4):255-62. · 1.42 Impact Factor
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ABSTRACT: We describe differential interference contrast (DIC) polarization anisotropy for tracking rotational dynamics of gold nanorod (AuNR) probes. DIC polarization anisotropy enabled us to reveal the unidirectional clockwise circular translocation of an AuNR attached to a kinesin-driven microtubule and to precisely determine the real-time orientation of the AuNR during the dynamic process.
Chemical Communications 07/2011; 47(27):7743-5. · 6.17 Impact Factor
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ABSTRACT: Superlocalization of single molecules and nanoparticles with a precision of subnanometer to a few tens of nanometers is crucial for elucidating nanoscale structures and movements in biological and chemical systems. A novel design of ultraflat and ultrathin glass/polydimethylsiloxane (PDMS) hybrid microdevices is introduced to provide almost uncompromised optical imaging quality for on-chip superlocalization and super-resolution imaging of single molecules and nanoparticles under a variety of microscopy modes. The performance of the high-fidelity (Hi-Fi) optical imaging microfluidic device was validated by precisely mapping micronecklaces made of fluorescent microtubules and 40 nm gold nanoparticles and by demonstrating the activation and excitation cycles of single Alexa Fluor 647 dyes for direct stochastic optical reconstruction microscopy in PDMS-based microchannels for the first time. Furthermore, the microdevice's feasibility for multimodality microscopy imaging was demonstrated by a vertical scan of live cells in epi-fluorescence and differential interference contrast (DIC) microscopy modes simultaneously.
Analytical Chemistry 06/2011; 83(13):5073-7. · 5.86 Impact Factor
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ABSTRACT: Pancreatic cancer (PC) is one of the most lethal malignancies. It has a 5-year survival rate of only 6%, owing in part to the lack of a reliable tumor marker for early diagnosis. Recent research has shown that the mucin protein MUC4 is aberrantly expressed in pancreatic adenocarcinoma cell lines and tissues but is undetectable in normal pancreas and chronic pancreatitis. Thus, the level of MUC4 in patient sera has the potential to function as a diagnostic and prognostic marker for PC. However, the measurement of MUC4 in sera using conventional test platforms (e.g., enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)) has been unsuccessful. This has prevented the assessment of the utility of this protein as a possible PC marker in sera. In addressing this obstacle, the work herein examines the potential to create a simple diagnostic test for MUC4 through the development of a surface-enhanced Raman scattering (SERS)-based immunoassay, which was then used to demonstrate the first ever detection of MUC4 in cancer patient serum samples. Importantly, these measurements showed that sera from patients with PC produced a significantly higher SERS response for MUC4 compared to sera from healthy individuals and from patients with benign diseases. These results indicate that a SERS-based immunoassay can monitor MUC4 levels in patient sera, representing a much needed first step toward assessing the potential of this protein to serve as a serum marker for the early stage diagnosis of PC. This paper details these and other findings (i.e., the detection of the mucin protein CA19-9), which demonstrate that our SERS assay outperforms conventional assays (i.e., RIA and ELISA) with respect to limits of detection, readout time, and required sample volume.
03/2011;
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ABSTRACT: Engineered nanoparticles have emerged as potentially revolutionary drug and gene delivery vectors. Using rod-shaped gold nanoparticles as a model, we studied for the first time the rotational dynamics of nanoparticle vectors on live cell membranes and its impact on the fate of these nanoparticle vectors. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at 200 frames/s under a differential interference contrast microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charges. Specific surface functional groups and the availability of receptors on cell membranes also contribute to the rotational dynamics. The study of rotational brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances.
Journal of the American Chemical Society 03/2011; 133(15):5720-3. · 9.91 Impact Factor
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ABSTRACT: Pancreatic cancer (PC) is one of the most lethal malignancies. It has a 5-year survival rate of only 6%, owing in part to the lack of a reliable tumor marker for early diagnosis. Recent research has shown that the mucin protein MUC4 is aberrantly expressed in pancreatic adenocarcinoma cell lines and tissues but is undetectable in normal pancreas and chronic pancreatitis. Thus, the level of MUC4 in patient sera has the potential to function as a diagnostic and prognostic marker for PC. However, the measurement of MUC4 in sera using conventional test platforms (e.g., enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)) has been unsuccessful. This has prevented the assessment of the utility of this protein as a possible PC marker in sera. In addressing this obstacle, the work herein examines the potential to create a simple diagnostic test for MUC4 through the development of a surface-enhanced Raman scattering (SERS)-based immunoassay, which was then used to demonstrate the first ever detection of MUC4 in cancer patient serum samples. Importantly, these measurements showed that sera from patients with PC produced a significantly higher SERS response for MUC4 compared to sera from healthy individuals and from patients with benign diseases. These results indicate that a SERS-based immunoassay can monitor MUC4 levels in patient sera, representing a much needed first step toward assessing the potential of this protein to serve as a serum marker for the early stage diagnosis of PC. This paper details these and other findings (i.e., the detection of the mucin protein CA19-9), which demonstrate that our SERS assay outperforms conventional assays (i.e., RIA and ELISA) with respect to limits of detection, readout time, and required sample volume.
Analytical Chemistry 03/2011; 83(7):2554-61. · 5.86 Impact Factor
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ABSTRACT: As noble metal nanoparticles are deployed into increasingly sophisticated environments, it is necessary to fully develop our understanding of nanoparticle behavior and the corresponding instrument responses. In this paper, we report on the optical response of three important gold nanorod configurations under dark field and differential interference contrast (DIC) microscopy after first establishing their absolute geometries with transmission electron microscopy (TEM). The observed longitudinal plasmon wavelengths of single nanorods are located at wavelengths consistent with previously developed theory. A dimer is shown exhibiting a multipole plasmon at wavelengths that are consistent with the dipole plasmon of single nanorods in the sample. DIC can also distinguish a single nanorod from a pair of uncoupled nanorods with an interparticle distance below the diffraction limit. The experimental observations are consistent with simulated DIC images using a DIC point spread function. The findings herein are a critical step toward being able to characterize nanorods in dynamic environments without the use of electron microscopy.
ACS Nano 12/2010; 4(12):7667-75. · 10.77 Impact Factor
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ABSTRACT: Gold nanorods are excellent orientation probes due to their anisotropic optical properties. Their dynamic rotational motion in the 3D space can be disclosed with Nomarski-type differential interference contrast (DIC) microscopy. We demonstrate that by using the combination of gold nanorod probes and DIC microscopy, we are able to resolve rotational motions of nano-cargos transported by motor proteins at video rate not only on engineered surfaces but also on cytoskeleton tracks in live cells.
Journal of the American Chemical Society 11/2010; 132(46):16417-22. · 9.91 Impact Factor
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ABSTRACT: The wavelength dependence of plasmonic nanoparticles' contrasts in differential interference contrast (DIC) microscopy has been exploited previously for unambiguous identification and dynamic tracking of these nanoprobes in complex environments (Anal. Chem. 2009, 81, 9203-9208). In the present study, the suitability of multiplexing detection in DIC microscopy was investigated systematically with 19 kinds of nanoparticles of different materials and/or sizes. A unique DIC contrast spectrum was found for each kind of nanoparticle. Multiplexing detection was accomplished by measuring DIC contrasts at a minimum of two specific illumination wavelengths. The main advantages of DIC microscopy for multiplexing detection over other nonfluorescence techniques, such as dark field microscopy and surface-enhanced Raman scattering, were demonstrated by differentiating four kinds of nanoparticles on the cell membrane while providing high-contrast images of both the nanoprobes and cell features.
Analytical Chemistry 08/2010; 82(15):6675-9. · 5.86 Impact Factor
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ABSTRACT: Precise imaging of cellular and subcellular structures and dynamic processes in live cells is crucial for fundamental research in life sciences and in medical applications. Non-fluorescent nanoparticles are an important type of optical probe used in live-cell imaging due to their photostability, large optical cross-sections, and low toxicity. Here, we provide an overview of recent developments in the optical imaging of non-fluorescent nanoparticle probes in live cells.
The Analyst 02/2010; 135(2):215-21. · 4.23 Impact Factor
