重要論文精選
  • Yi-Hung Liao, Chih-Hsiang Lin, Ching-Ya Cheng, Wai Cheng Wong, Jz-Yuan Juo, Chia-Lung Hsieh*, ACS Nano 13(10), 10918–10928 (2019) (重要論文精選) Website
    Monovalent and oriented labeling of gold nanoprobes for the high-resolution tracking of a single membrane molecule
    Abstract
    Single-molecule tracking is a powerful method to study molecular dynamics in living systems including biological membranes. High-resolution single-molecule tracking requires a bright and stable signal, which has typically been facilitated by nanoparticles due to their superb optical properties. However, there are concerns about using a nanoparticle to label a single molecule because of its relatively large size and the possibility of crosslinking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects of the use of nanoparticles to measure the diffusion of single membrane molecules. By conjugating a low density of streptavidin (sAv) to gold nanoparticles (AuNPs) of different sizes (10, 15, 20, 30, and 40 nm), we isolate and quantify the effect of the particle size on the diffusion of biotinylated lipids in supported lipid bilayers (SLBs). We find that single sAv tends to crosslink two biotinylated lipids, leading to a much slower diffusion in SLBs. We further demonstrate a simple and robust strategy for the monovalent and oriented labeling of a single lipid molecule with a AuNP by using naturally dimeric rhizavidin (rAv) as a bridge, thus connecting the biotinylated nanoparticle surface and biotinylated target molecule. The rAv-AuNP conjugate demonstrates fast and free diffusion in SLBs (2–3 μm2/s for rAv-AuNP sizes of 10 nm to 40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and crosslinking effects are successfully avoided. We also note that the diffusion of dye-labeled lipids critically depends on the choice of dye, which could report different diffusion coefficients by about 20 % (2.2 μm2/s of ATTO647N and 2.6 μm2/s of ATTO532). By comparing the diffusion of the uniformly and randomly oriented labeling of a single lipid molecule with a AuNP, we conclude that oriented labeling is favorable for measuring the diffusion of single membrane molecules. Our work shows that the measured diffusion of the membrane molecule is highly sensitive to the molecular design of the crosslinker for labeling. The demonstrated approach of monovalent and oriented AuNP labeling provides the opportunity to study single molecule membrane dynamics at much higher spatiotemporal resolutions, and most importantly, without labeling artifacts.
  • Ching-Ya Cheng, Yi-Hung Liao, Chia-Lung Hsieh*, Nanoscale 11, 568–577 (2019) (重要論文精選) Website
    High-speed imaging and tracking of very small single nanoparticles by contrast enhanced microscopy
    Abstract
    Nanoparticles have been used extensively in biology-related research and many applications require direct visualization of individual nanoparticles under optical microscopy. For long-term and high-speed measurements, scattering-based microscopy is a unique technique because of the stable and indefinite scattering signal. In scattering-based single-particle measurements, large nanoparticles are usually needed in order to generate sufficient signal for detection. However, larger nanoparticle introduces greater mass loading, experiences stronger steric hindrance, and is more prone to crosslinking. In this work, we demonstrate coherent brightfield (COBRI) microscopy with enhanced contrast and show its capability of direct visualization of very small nanoparticles in scattering at high speed. The COBRI microscopy allows us to visualize and track single metallic and dielectric nanoparticles, as small as 10 nm, at 1,000 frames per second. A quantitative relationship between the linear scattering cross section of nanoparticle and its COBRI contrast is reported. Using COBRI microscopy, we further demonstrate tracking of 10 nm gold nanoparticles labeled to lipid molecules in supported bilayer membranes, showing that the small nanoparticle may facilitate single-molecule measurements with reduced perturbation. Furthermore, identical imaging sensitivity of COBRI and interferometric scattering (iSCAT) microscopy, the reflection counterpart of COBRI, is demonstrated under equal illumination intensity. Finally, future improvements in speed and sensitivity of scattering-based interference microscope are discussed.
  • Yi-Fan Huang, Guan-Yu Zhuo, Chun-Yu Chou, Cheng-Hao Lin, Wen Chang, and Chia-Lung Hsieh*, ACS Nano 11(3), 2575–2585 (2017) (重要論文精選) Website
    Coherent brightfield microscopy provides the spatiotemporal resolution to study early stage viral infection in live cells
    Abstract
    Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early-stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (< 3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image post-processing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (~1 μm2/s) at microsecond timescales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.
  • Ching-Ya Cheng and Chia-Lung Hsieh*, ACS Photonics 4(7), 1730–1739 (2017) (重要論文精選) Website
    Background estimation and correction for high-precision localization microscopy
    Abstract
    Localization of a single nanosized light emitter has substantial applications in bioimaging. The accuracy and precision of localization are limited by the noise and the heterogeneous background superimposed on the signal. While the effects of noise are well recognized, the influence of background is less addressed. Proper background correction not only provides more accurate localization data but also enhances the sensitivity of detection. Here, we demonstrate a new approach to background correction by estimating and removing the heterogeneous but stationary background from a series of images containing a spatially moving signal. Our approach exploits the correlated signal information encoded in the neighboring pixels governed by the point-spread function of the measurement system. This new approach makes it possible to obtain the background even when the total displacement of the signal is subdiffraction limited throughout the observation, the scenario where previous methods become invalid. We characterize our approach systematically with different types of signal motions at various signal-to-noise ratios in numerical simulations. We then verify our method experimentally by recovering the nanoscopic displacements of single gold nanoparticle moving in a specified pattern and a single virus particle randomly diffusing on a cell surface. The source code of our algorithm written in MATLAB is provided together with a sample data set. Our approach has immediate applications in high-precision optical localization measurements.
  • Hsiao-Mei Wu, Ying-Hsiu Lin, Tzu-Chi Yen, and Chia-Lung Hsieh*, Scientific Reports 6 20542 (2016) (重要論文精選) Website
    Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking
    Abstract
    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.
2019
  • Yi-Hung Liao, Chih-Hsiang Lin, Ching-Ya Cheng, Wai Cheng Wong, Jz-Yuan Juo, Chia-Lung Hsieh*, ACS Nano 13(10), 10918–10928 (2019) Website
    Monovalent and oriented labeling of gold nanoprobes for the high-resolution tracking of a single membrane molecule
    Abstract
    Single-molecule tracking is a powerful method to study molecular dynamics in living systems including biological membranes. High-resolution single-molecule tracking requires a bright and stable signal, which has typically been facilitated by nanoparticles due to their superb optical properties. However, there are concerns about using a nanoparticle to label a single molecule because of its relatively large size and the possibility of crosslinking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects of the use of nanoparticles to measure the diffusion of single membrane molecules. By conjugating a low density of streptavidin (sAv) to gold nanoparticles (AuNPs) of different sizes (10, 15, 20, 30, and 40 nm), we isolate and quantify the effect of the particle size on the diffusion of biotinylated lipids in supported lipid bilayers (SLBs). We find that single sAv tends to crosslink two biotinylated lipids, leading to a much slower diffusion in SLBs. We further demonstrate a simple and robust strategy for the monovalent and oriented labeling of a single lipid molecule with a AuNP by using naturally dimeric rhizavidin (rAv) as a bridge, thus connecting the biotinylated nanoparticle surface and biotinylated target molecule. The rAv-AuNP conjugate demonstrates fast and free diffusion in SLBs (2–3 μm2/s for rAv-AuNP sizes of 10 nm to 40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and crosslinking effects are successfully avoided. We also note that the diffusion of dye-labeled lipids critically depends on the choice of dye, which could report different diffusion coefficients by about 20 % (2.2 μm2/s of ATTO647N and 2.6 μm2/s of ATTO532). By comparing the diffusion of the uniformly and randomly oriented labeling of a single lipid molecule with a AuNP, we conclude that oriented labeling is favorable for measuring the diffusion of single membrane molecules. Our work shows that the measured diffusion of the membrane molecule is highly sensitive to the molecular design of the crosslinker for labeling. The demonstrated approach of monovalent and oriented AuNP labeling provides the opportunity to study single molecule membrane dynamics at much higher spatiotemporal resolutions, and most importantly, without labeling artifacts.
  • Wai Cheng Wong, Jz-Yuan Juo, Yi-Hung Liao, Ching-Ya Cheng, Chih-Hsiang Lin, Chia-Lung Hsieh*, Journal of Physical Chemistry B 123 (30), 6492–6504   (2019) Website
    Characterization of single-protein dynamics in polymer-cushioned lipid bilayers derived from cell plasma membranes
    Abstract
    Native cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm2/s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer cushioned plasma membrane bilayers that are potentially useful for future developments of membrane-based biosensors and analytical assays.
  • Ching-Ya Cheng, Yi-Hung Liao, Chia-Lung Hsieh*, Nanoscale 11, 568–577 (2019) Website
    High-speed imaging and tracking of very small single nanoparticles by contrast enhanced microscopy
    Abstract
    Nanoparticles have been used extensively in biology-related research and many applications require direct visualization of individual nanoparticles under optical microscopy. For long-term and high-speed measurements, scattering-based microscopy is a unique technique because of the stable and indefinite scattering signal. In scattering-based single-particle measurements, large nanoparticles are usually needed in order to generate sufficient signal for detection. However, larger nanoparticle introduces greater mass loading, experiences stronger steric hindrance, and is more prone to crosslinking. In this work, we demonstrate coherent brightfield (COBRI) microscopy with enhanced contrast and show its capability of direct visualization of very small nanoparticles in scattering at high speed. The COBRI microscopy allows us to visualize and track single metallic and dielectric nanoparticles, as small as 10 nm, at 1,000 frames per second. A quantitative relationship between the linear scattering cross section of nanoparticle and its COBRI contrast is reported. Using COBRI microscopy, we further demonstrate tracking of 10 nm gold nanoparticles labeled to lipid molecules in supported bilayer membranes, showing that the small nanoparticle may facilitate single-molecule measurements with reduced perturbation. Furthermore, identical imaging sensitivity of COBRI and interferometric scattering (iSCAT) microscopy, the reflection counterpart of COBRI, is demonstrated under equal illumination intensity. Finally, future improvements in speed and sensitivity of scattering-based interference microscope are discussed.
  • Chia-Lung Hsieh, book chapter, Springer, ISBN 978-3-030-21722-8 (2019) Website
    Label-free, ultrahigh-speed, direct imaging and tracking of bionanoparticles in live cells by using coherent brightfield microscopy
    Abstract
    Many important biological phenomena, ranging from cell signaling to viral infection, are facilitated by transportation of biological substances encapsulated in native nano-sized particles. Thermal fluctuation drives nanoparticles through cellular environments; this movement is facilitated by their small size. To understand how a specific cell function can be achieved through random collisions, it is useful to know the interactions between single particles and the local environment, as determined by measuring cell dynamics at high spatial and temporal resolutions. In this chapter, a simple yet powerful wide-field optical technique, coherent brightfield (COBRI) microscopy, is presented. COBRI microscopy detects linearly scattered light from a nanoparticle through imaging-based interferometry, which enables direct observation of small biological nanoparticles in live cells without labels. Proper image post-processing further improves the detection sensitivity of small particles by removing the scattering background of cell structures. COBRI microscopy can easily operate at a high speed due to its wide-field nature and stable, indefinite scattering signal. Using COBRI, the dynamics of single virus particles and cell vesicles in live cells can be successfully captured at a microsecond temporal resolution and nanometer spatial precision in three dimensions. The ultrahigh spatiotemporal resolution and shot-noise-limited sensitivity of COBRI microscopy provide an opportunity to study the biophysics and biochemistry of live cells at the nanoscale.
  • Feng-Jen Hsieh, Shingo Sotoma, Hsin-Hung Lin, Ching-Ya Cheng, Tsyr-Yan Yu, Chia-Lung Hsieh, Chun-Hung Lin*, Huan-Cheng Chang*, ACS Applied Materials & Interfaces 11(22), 19774-19781 (2019) Website
    Bioorthogonal fluorescent nanodiamonds for continuous long-term imaging and tracking of membrane proteins
    Abstract
    Real-time tracking of membrane proteins is essential to gain an in-depth understanding of their dynamics on cell surface. However, con-ventional fluorescence imaging with molecular probes like organic dyes and fluorescent proteins often suffers from photobleaching of the fluorophores, thus hindering their use for continuous long-term observations. With the availability of fluorescent nanodiamonds (FNDs), which have superb biocompatibility and excellent photostability, it is now possible to conduct the imaging in both short and long terms with high temporal and spatial resolution. To realize the concept, we have developed a facile method (e.g., one-pot preparation) to produce alkyne-functionalized hyperbranched-polyglycerol-coated FNDs for bioorthogonal labelling of azide-modified membrane proteins and azide-modified antibodies of membrane proteins. The high specificity of this labelling method has allowed us to continuously monitor the movements of the proteins of interest (such as integrin α5) on/in living cells over 2 h. The results open a new horizon for live cell imaging with functional nanoparticles and fluorescence microscopy.
2018
  • Edward Lyman*, Chia-Lung Hsieh, Christian Eggeling, Biophysical Journal 115, 595–604 (2018) Website
    From dynamics to membrane organization: Experimental breakthroughs occasion a "modeling manifesto"
    Abstract
    New experimental techniques especially in the context of observing molecular dynamics reveal the plasma membrane to be heterogeneous and "scale-rich," from nanometers to microns, and from microseconds to seconds. This is critical information, as heterogeneous, scale-dependent transport governs the molecular encounters that underlie cellular signaling. The data are rich, and reaffirm the importance of the cortical cytoskeleton, protein aggregates, and lipidomic complexity to the statistics of molecular encounters. Moreover, the data demand simulation approaches with a particular set of features, hence the “manifesto”. Together with the experimental data, simulations which satisfy these requirements hold the promise of a deeper understanding of membrane spatiotemporal organization. Several experimental breakthroughs in measuring molecular membrane dynamics are reviewed, the constraints that they place on simulations are discussed, and the status of simulation approaches which aim to meet them are detailed.
  • Chia-Lung Hsieh*, Optics Communications 422, 69–74 (2018) Website
    Label-free, ultrasensitive, ultrahigh-speed scattering-based interferometric imaging
    Abstract
    Label-free microscope imaging techniques allow direct visualization of biological substances in their most native forms. This review article provides an overview of recent advancements of scattering-based, interferometric laser microscopy and their applications to ultrasensitive and ultrahigh-speed biological imaging. In particular, common-path, widefield interferometric laser microscopy, namely interferometric scattering (iSCAT) microscopy and coherent brightfield (COBRI) microscopy, are discussed in details. Using these simple yet powerful optical techniques, single proteins and individual endogenous biological nanoparticles can be imaged and tracked without any label at ultrahigh spatiotemporal resolution. The development of ultrasensitive and ultrahigh-speed scattering-based interferometric microscope imaging enables investigation of biophysical and biochemical processes with minimal perturbation and unprecedented clarity.
2017
  • Ching-Ya Cheng and Chia-Lung Hsieh*, ACS Photonics 4(7), 1730–1739 (2017) Website
    Background estimation and correction for high-precision localization microscopy
    Abstract
    Localization of a single nanosized light emitter has substantial applications in bioimaging. The accuracy and precision of localization are limited by the noise and the heterogeneous background superimposed on the signal. While the effects of noise are well recognized, the influence of background is less addressed. Proper background correction not only provides more accurate localization data but also enhances the sensitivity of detection. Here, we demonstrate a new approach to background correction by estimating and removing the heterogeneous but stationary background from a series of images containing a spatially moving signal. Our approach exploits the correlated signal information encoded in the neighboring pixels governed by the point-spread function of the measurement system. This new approach makes it possible to obtain the background even when the total displacement of the signal is subdiffraction limited throughout the observation, the scenario where previous methods become invalid. We characterize our approach systematically with different types of signal motions at various signal-to-noise ratios in numerical simulations. We then verify our method experimentally by recovering the nanoscopic displacements of single gold nanoparticle moving in a specified pattern and a single virus particle randomly diffusing on a cell surface. The source code of our algorithm written in MATLAB is provided together with a sample data set. Our approach has immediate applications in high-precision optical localization measurements.
  • Yi-Fan Huang, Guan-Yu Zhuo, Chun-Yu Chou, Cheng-Hao Lin, Wen Chang, and Chia-Lung Hsieh*, ACS Nano 11(3), 2575–2585 (2017) Website
    Coherent brightfield microscopy provides the spatiotemporal resolution to study early stage viral infection in live cells
    Abstract
    Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early-stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (< 3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image post-processing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (~1 μm2/s) at microsecond timescales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.
  • Yi-Fan Huang, Guan-Yu Zhuo, Chun-Yu Chou, Cheng-Hao Lin, and Chia-Lung Hsieh*, Nanoscale 9(19), 6567–6574 (2017) Website
    Label-free, ultrahigh-speed, 3D observation of bidirectional and correlated intracellular cargo transport by coherent brightfield microscopy
    Abstract
    Investigation of intracellular transport at the molecular scale requires measurements at high spatial and temporal resolutions. We demonstrate label-free, direct imaging and tracking of native cell vesicles in live cells at ultrahigh spatiotemporal resolution. Using coherent brightfield (COBRI) microscopy, we monitor individual cell vesicles traveling inside the cell with nanometer spatial precision in 3D at 30,000 frames per second. Stepwise directional motion of the vesicle on the cytoskeletal track is clearly resolved. We also observe repeated switching of transport direction of the vesicle in a continuous trajectory. Our high-resolution measurement unveils transient pausing and subtle bidirectional motion of the vesicle, taking place over tens of nanometers in tens of milliseconds. By tracking multiple particles simultaneously, we found strong correlations between the motions of two neighboring vesicles. Our label-free ultrahigh-speed optical imaging provides the opportunity to visualize intracellular cargo transport at the nanoscale in the microsecond timescale with minimal perturbation.
  • Minh D. Pham, Chandra Prakash Epperla, Chia-Lung Hsieh, Wen Chang, and Huan-Cheng Chang*, Analytical Chemistry 89(12), 6527–6534 (2017) Website
    Glycosaminoglycans-specific cell targeting and imaging using fluorescent nanodiamonds coated with viral envelope proteins
    Abstract
    Understanding virus-host interactions is crucial for vaccine development. This study investigates such interactions using fluorescent nanodiamonds (FNDs) coated with vaccinia envelope proteins as the model system. To achieve this goal, we noncovalently conjugated 100-nm FND with A27(aa 21–84), a recombinant envelope protein of vaccinia virus, for glycosaminoglycans (GAGs)-specific targeting and imaging of living cells. Another recombinant protein A27(aa 33–84) that removes the GAGs-binding sequences was also used for comparison. Three types of A27-coated FNDs were generated, including A27(aa 21–84)-FND, A27(aa 33–84)-FND, and hybrid A27(aa 21–84)/A27(aa 33–84)-FND. The specificity of these viral protein-FND conjugates toward GAGs binding was examined by flow cytometry, fluorescence microscopy, and gel electrophoresis. Results obtained for normal and GAGs-deficient cells showed that the recombinant proteins maintain their GAG-targeting activities even after immobilization on the FND surface. Our studies provide a new nanoparticle-based platform not only to target specific cell types, but also to track the fates of these immobilized viral proteins in targeted cells as well as to isolate and enrich GAGs associated proteins on cell membrane.
  • Chia-Lung Hsieh*, SPIE Newsroom (2017) Website
    Ultrahigh-speed imaging reveals nanoscopic single-molecule dynamics
    Abstract
    The journey of a single molecule can be followed with nanometer spatial precision and microsecond temporal resolution by using interferometric scattering optical microscopy and an ultrahigh-speed camera.
2016
  • Jeong Min Lee, Jung A. Kim, Tzu-Chi Yen, In Hwan Lee, Byungjun Ahn, Younghoon Lee, Chia-Lung Hsieh, Ho Min Kim, and Yongwon Jung, Angewandte Chemie 128(10), 3454–3458 (2016) Website
    A rhizavidin monomer with nearly multimeric avidin-like binding stability against biotin conjugates
    Abstract
    Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein—enhanced monoavidin—with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells.
  • Hsiao-Mei Wu, Ying-Hsiu Lin, Tzu-Chi Yen, and Chia-Lung Hsieh*, Scientific Reports 6 20542 (2016) Website
    Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking
    Abstract
    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.
2014
  • Chia-Lung Hsieh, Susann Spindler, Jens Ehrig, and Vahid Sandoghdar*, Journal of Physical Chemistry B 118(6), 1545–1554 (2014) Website
    Tracking single particles on supported lipid membranes: multimobility diffusion and nanoscopic confinement
    Abstract
    Supported lipid bilayers have been studied intensively over the past two decades. In this work, we study the diffusion of single gold nanoparticles (GNPs) with diameter of 20 nm attached to GM1 ganglioside or DOPE lipids at different concentrations in supported DOPC bilayers. The indefinite photostability of GNPs combined with the high sensitivity of interferometric scattering microscopy (iSCAT) allows us to achieve 1.9 nm spatial precision at 1 ms temporal resolution, while maintaining long recording times. Our trajectories visualize strong transient confinements within domains as small as 20 nm, and the statistical analysis of the data reveals multiple mobilities and deviations from normal diffusion. We present a detailed analysis of our findings and provide interpretations regarding the effect of the supporting substrate and GM1 clustering. We also comment on the use of high-speed iSCAT for investigating diffusion of lipids, proteins, or viruses in lipid membranes with unprecedented spatial and temporal resolution.
  • Ying-Hsiu Lin, Wei-Lin Chang, and Chia-Lung Hsieh*, Optics Express 22(8), 9159–9170 (2014) Website
    Shot-noise limited localization of single 20 nm gold particles with nanometer spatial precision within microseconds
    Abstract
    Single-particle tracking (SPT) is a powerful approach to investigate dynamics without ensemble average. Continuing effort has been made to track smaller particles with better spatial precision at higher speed. In this work, we demonstrate SPT of 20 nm gold nanoparticle (GNP) with 2 nm spatial precision up to 500 kHz by using microsecond interferometric scattering (μs-iSCAT) microscopy. The linear scattering signal from single GNPs is detected by a high-speed CMOS camera via interference. Through this homodyne detection, shot-noise limited sensitivity, and therefore optimal localization precision are achieved at high speed where considerable electronic noise is present. Using μs-iSCAT microscopy, we observe anomalous diffusion of GNPs labeled to lipid molecules in a supported bilayer membrane prepared on a glass substrate. The combination of nanometer spatial precision and microsecond temporal resolution provides the opportunity to study rapid motions of nano-objects on molecular scale with unprecedented clarity.
2012
  • Xin Yang, Ye Pu, Chia-Lung Hsieh, Cheng Ai Ong, Demetri Psaltis, and Konstantina M. Stankovicc, Journal of Biomedical Optics 18(3), 031104 (2012)
    Two-photon microscopy of the mouse cochlea in situ for cellular diagnosis
    Abstract
  • Xin Yang, Chia-Lung Hsieh, Ye Pu, and Demetri Psaltis, Optics Express 20(3), 2500–2506 (2012) Website
    Three-dimensional scanning microscopy through thin turbid media.
    Abstract
    We demonstrate three-dimensional imaging through a thin turbid medium using digital phase conjugation of the second harmonic signal emitted from a beacon nanoparticle. The digitally phase-conjugated focus scans the volume in the vicinity of its initial position through numerically manipulated phase patterns projected onto the spatial light modulator. Accurate three dimensional images of a fluorescent sample placed behind a turbid medium are obtained.
2011
  • Rachel Grange, Thomas Lanvin, Chia-Lung Hsieh, Ye Pu, and Demetri Psaltis, Biomedical Optics Express 2(9), 2532–2539 (2011) Website
    Imaging with second-harmonic radiation probes in living tissue.
    Abstract
    We demonstrate that second-harmonic radiation imaging probes are efficient biomarkers for imaging in living tissue. We show that 100 nm and 300 nm BaTiO(3) nanoparticles used as contrast markers could be detected through 50 $μ$m and 120 $μ$m of mouse tail tissue in vitro or in vivo. Experimental results and Monte-Carlo simulations are in good agreement.
2010
  • Ye Pu, Rachel Grange, Chia-Lung Hsieh, and Demetri Psaltis, Physical Review Letters 104(20), 207402 (2010) Website
    Nonlinear Optical Properties of Core-Shell Nanocavities for Enhanced Second-Harmonic Generation
    Abstract
    A nonlinear optical plasmonic core-shell nanocavity is demonstrated as an efficient, subwavelength coherent light source through second-harmonic generation. The nonlinear optical plasmonic nanocavity incorporates a noncentrosymmetric medium, which utilizes the entire mode volume for even-order nonlinear optical processes. In previous plasmonic nanocavities, enhancement of such processes was only possible at the interface but symmetry prohibited in the body. We measured an enhancement of over 500 times in the second-harmonic radiation power. Calculations show that an enhancement of over 3500 times is achievable.
  • Chia-Lung Hsieh*, Ye Pu, Rachel Grange, and Demetri Psaltis, Optics Express 18(12),12283–12290 (2010) Website
    Digital phase conjugation of second harmonic radiation emitted by nanoparticles in turbid media
    Abstract
    We demonstrate focusing coherent light on a nanoparticle through turbid media based on digital optical phase conjugation of second harmonic generation (SHG) field from the nanoparticle. A SHG active nanoparticle inside a turbid medium was excited at the fundamental frequency and emitted SHG field as a point source. The SHG emission was scattered by the turbid medium, and the scattered field was recorded by off-axis digital holography. A phase-conjugated beam was then generated by using a phase-only spatial light modulator and sent back through the turbid medium, which formed a nearly ideal focus on the nanoparticle.
  • Chia-Lung Hsieh*, Rachel Grange, Ye Pua, Demetri Psaltis, Biomaterials 31(8), 2272–2277 (2010) Website
    Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes.
    Abstract
    The second harmonic generation (SHG) active nanocrystals have been demonstrated as attractive imaging probes in nonlinear microscopy due to their coherent, non-bleaching and non-blinking signals with a broad flexibility in the choice of excitation wavelength. For the use of these nanocrystals as biomarkers, it is essential to prepare a chemical interface for specific labeling. We developed a specific labeling scheme for barium titanate (BaTiO3) nanocrystals which we use as second harmonic radiation imaging probes. The specificity was achieved by covalently coupling antibodies onto the nanocrystals. We demonstrate highly specific labeling of the nanocrystal conjugates in an antibody microarray and also the membrane proteins of live biological cells in vitro. The development of surface functionalization and bioconjugation of SHG active nanocrystals provides the opportunities of applying them to biological studies.
  • Chia-Lung Hsieh*, Ye Pu, Rachel Grange, and Demetri Psaltis, Optics Express 18(11), 11917–11932 (2010) Website
    Second harmonic generation from nanocrystals under linearly and circularly polarized excitations.
    Abstract
    We study second harmonic generation (SHG) from non-centrosymmetric nanocrystals under linearly polarized (LP) and circularly polarized (CP) excitations. Theoretical models are developed for SHG from nanocrystals under both plane-wave and focused excitations. We find that the focused excitation reduces the polarization dependency of the SHG signal. We show that the SHG response under CP excitation is generally inferior to the average of LP excitations over all orientations. We verify the theory by measuring the SHG polar responses from BaTiO3 nanocrystals with a scanning confocal microscope. The experimental data agrees well with the theory.
  • Chia-Lung Hsieh*, Ye Pu, Rachel Grange, Grégoire Laporte, and Demetri Psaltis, Optics Express 18(20), 20723–20731 (2010) Website
    Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle.
    Abstract
    We demonstrate imaging through a turbid layer by using digital phase conjugation of the second harmonic field radiated from a beacon nanoparticle. We show that the phase-conjugated focus can be displaced from its initial position by illuminating the same region of the turbid layer with an angular offset. An image is obtained by scanning the phase-conjugated focus through the turbid layer in a region around the nanoparticle. We obtain a clear image of the target by measuring the light transmitted through it when scanning the focused beam.
2009
  • Chia-Lung Hsieh*, Rachel Grange, Ye Pu, and Demetri Psaltis, Optics Express 17(4), 2880–2891 (2009) Website
    Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging.
    Abstract
    Luminescent markers play a key role in imaging techniques for life science since they provide a contrast mechanism between signal and background. We describe a new type of marker using second harmonic generation (SHG) from noncentrosymmetric BaTiO(3) nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and coherent signal with a femtosecond-scale response time and broad flexibility in the choice of excitation wavelength. We obtained monodispersed BaTiO(3) nanoparticles in colloidal suspensions by coating the particle surface with amine groups. We characterized the SHG efficiency of 90-nm BaTiO(3) particles experimentally and theoretically. Moreover, we use the coherent SHG signal from BaTiO(3) nanoparticles for three-dimensional (3D) imaging without scanning. We built a harmonic holographic (H(2)) microscope which records digital holograms at the second harmonic frequency. For the first time, high-resolution 3D distributions of these SHG markers in mammalian cells are successfully captured and interpreted by the H(2) microscope.
  • Yu-Chieh Wen, Chia-Lung Hsieh, Kung-Hsuan Lin, Hung-Pin Chen, Shu-Cheng Chin, Ching-Lien Hsiao, Yuan-Ting Lin, Chia-Seng Chang, Yuan-Chih Chang, Li-Wei Tu, and Chi-Kuang Sun, Physical Review Letters 103(26), 264301 (2009) Website
    Specular scattering probability of acoustic phonons in atomically flat interfaces
    Abstract
    We report a direct determination of the specular scattering probability of acoustic phonons at a crystal boundary by observing the escape of incident coherent phonons from the coherent state during reflection. In the sub-THz frequency range where the phonon wavelength is much longer than the lattice constant, the acoustic phonon-interface interaction is found to agree well with the macroscopic theory on wave scattering from rough surfaces. This examination thus quantitatively verifies the dominant role of atomic-scale corrugations in the Kapitza anomaly observed at 1–10 K and further opens a new path to nondestructively estimate subnanoscale roughness of buried interfaces.
  • Rachel Grange, Jae-Woo Choi, Chia-Lung Hsieh, Ye Pu, Arnaud Magrez, Rita Smajda, László Forró, and Demetri Psaltis, Applied Physics Letters 95(14), 143105 (2009) Website
    Lithium niobate nanowires synthesis, optical properties, and manipulation
    Abstract
    Free-standing lithium niobate nanowires (LiNbO3) are synthesized by the hydrothermal route. The polarization response of the second harmonic generation (SHG) signal is measured in a single nanowire and used to identify the crystal orientation by matching with bulk LiNbO3 nonlinear optical susceptibility. The electrical manipulation of a LiNbO3 nanowire and its monitoring through the SHG signal in a fluidic setup are demonstrated.
2005
  • Cheng-Ta Yu, Kung-Hsuan Lin, Chia-Lung Hsieh, Chang-Chi Pan, Jen-Inn Chyi, and Chi-Kuang Suna, Applied Physics Letters 87(9), 093114 (2005) Website
    Generation of frequency-tunable nanoacoustic waves by optical coherent control
    Abstract
    We have developed a system to generate arbitrary wave-form nanoacoustic waves (NAWs) with a piezoelectric InGaN∕GaN single-quantum well. Based on an optical coherent control technique, acoustic frequency tunability in the subterahertz range is realized within only one fixed sample. The acoustic generation mechanisms, especially the in-wel piezoelectric field Coulomb screening which tends to be saturated at high carrier concentrations, are discussed with optical power dependency. With the generated NAWs propagating in the c axis of a GaN thin film, the lifetime of the 500 GHz longitudinal-acoustic phonon pulses in GaN is measured to be longer than 420 ps, corresponding to a GaN depth more than 3.3μm.
2004
  • Chia-Lung Hsieh, Kung-Hsuan Lin, Shr-Bin Wu, Chang-Chi Pan, Jen-Inn Chyi, and Chi-Kuang Suna, Applied Physics Letters 85(20), 4735–4737 (2004) Website
    Reflection property of nano-acoustic wave at the air∕GaN interface
    Abstract
    We present a study on the total internal reflection of nano-acoustic waves (NAWs) at the air∕GaN interface. The coherent NAW was generated and detected by piezoelectric InGaN∕GaN multiple quantum wells using a femtosecond transient transmission technique. With suitably designed sample structures, the fact that strain of the NAW experiences a sign change after total internal reflection at the air∕solid interface is examined directly. The surface roughness of the sample was found to distort the wave front of the NAW and to diminish the measured amplitude of the reflected NAW.