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 postprocessing, 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 μm/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

We provide evidence that the chirality of collagen can give rise to strong second-harmonic generation circular dichroism (SHG-CD) responses in nonlinear microscopy. Although chirality is an intrinsic structural property of collagen, most of the previous studies ignore that property. We demonstrate chiral imaging of individual collagen fibers by using a laser scanning microscope and type-I collagen from pig ligaments. 100% contrast level of SHG-CD is achieved with sub-micrometer spatial resolution. As a new contrast mechanism for imaging chiral structures in bio-tissues, this technique provides information about collagen morphology and three-dimensional orientation of collagen molecules.

Chiral molecules are stereoselective with regard to specific biological functions. Enantiomers differ considerably in their physiological reactions with the human body. Safeguarding the quality and safety of drugs requires an efficient analytical platform by which to selectively probe chiral compounds to ensure the extraction of single enantiomers. Asymmetric synthesis is a mature approach to the production of single enantiomers; however, it is poorly suited to mass production and allows for only specific enantioselective reactions. Furthermore, it is too expensive and time-consuming for the evaluation of therapeutic drugs in the early stages of development. These limitations have prompted the development of surface-modified nanoparticles using amino acids, chiral organic ligands, or functional groups as chiral selectors applicable to a racemic mixture of chiral molecules. The fact that these combinations can be optimized in terms of sensitivity, specificity, and enantioselectivity makes them ideal for enantiomeric recognition and separation. In chiral resolution, molecules bond selectively to particle surfaces according to homochiral interactions, whereupon an enantiopure compound is extracted from the solution through a simple filtration process. In this review article, we discuss the fabrication of chiral nanoparticles and look at the ways their distinctive surface properties have been adopted in enantiomeric recognition and separation.

We show that scattering from a single gold nanoparticle is saturable for the first time. Wavelength-dependent study reveals that the saturation behavior is governed by depletion of surface plasmon resonance, not the thermal effect. We observed interesting flattening of the point spread function of scattering from a single nanoparticle due to saturation. By extracting the saturated part of scattering via temporal modulation, we achieve λ/8 point spread function in far-field imaging with unambiguous separation of adjacent particles.

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The investigation of intracellular transport at the molecular scale requires measurements at high spatial and temporal resolutions. We demonstrate the label-free, direct imaging and tracking of native cell vesicles in live cells at an 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. The stepwise directional motion of the vesicle on the cytoskeletal track is clearly resolved. We also observe the repeated switching of the transport direction of the vesicle in a continuous trajectory. Our high-resolution measurement unveils the 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.

SummaryChirality is one of the most fundamental and essential structural properties of biological molecules. Many important biological molecules including amino acids and polysaccharides are intrinsically chiral. Conventionally, chiral species can be distinguished by interaction with circularly polarized light, and circular dichroism is one of the best-known approaches for chirality detection. As a linear optical process, circular dichroism suffers from very low signal contrast and lack of spatial resolution in the axial direction. It has been demonstrated that by incorporating nonlinear interaction with circularly polarized excitation, second-harmonic generation circular dichroism can provide much higher signal contrast. However, previous circular dichroism and second-harmonic generation circular dichroism studies are mostly limited to probe chiralities at surfaces and interfaces. It is known that secondharmonic generation, as a second-order nonlinear optical effect, provides excellent optical sectioning capability when combined with a laser-scanning microscope. In this work, we combine the axial resolving power of second-harmonic generation and chiral sensitivity of second-harmonic generation circular dichroism to realize three-dimensional chiral detection in biological tissues. Within the point spread function of a tight focus, second-harmonic generation circular dichroism could arise from the macroscopic supramolecular packing as well as the microscopic intramolecular chirality, so our aim is to clarify the origins of second-harmonic generation circular dichroism response in complicated three-dimensional biological systems.Correspondence to: Shi-Wei Chu, 1, Sec 4, Roosevelt Rd., Taipei 10617, Taiwan. Tel: +88-623-366-5131; fax: +88-622-363-9984; e-mail: swchu@phys.ntu.edu.twThe sample we use is starch granules whose secondharmonic generation-active molecules are amylopectin with both microscopic chirality due to its helical structure and macroscopic chirality due to its crystallized packing. We found that in a starch granule, the second-harmonic generation for right-handed circularly polarized excitation is significantly different from second-harmonic generation for left-handed one, offering excellent second-harmonic generation circular dichroism contrast that approaches 100%. In addition, three-dimensional visualization of secondharmonic generation circular dichroism distribution with sub-micrometer spatial resolution is realized. We observed second-harmonic generation circular dichroism sign change across the starch granules, and the result suggests that in thick biological tissue, second-harmonic generation circular dichroism arises from macroscopic molecular packing. Our result provides a new method to visualize the organization of three-dimensional structures of starch granules. The second-harmonic generation circular dichroism imaging method expands the horizon of nonlinear chiroptical studies from simplified surface/solution environments to complicated biological tissues.

Non-linear optical (NLO) microscopy has proven to be a powerful tool especially for tissue imaging with sub-cellular resolution, high penetration depth, endogenous contrast specificity, pinhole-less optical sectioning capability. In this review, we discuss label-free non-linear optical microscopes including the two-photon fluorescence (TPF), fluorescence lifetime imaging microscopy (FLIM), polarization-resolved second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) techniques with various samples. The non-linear signals are generated from collagen in tissue (SHG), amylopectin from starch granules (SHG), sarcomere structure of fresh muscle (SHG), elastin in skin (TPF), nicotinamide adenine dinucleotide (NADH) in cells (TPF), and lipid droplets in cells (CARS). Again, the non-linear signals are very specific to the molecular structure of the sample and its relative orientation to the polarization of the incident light. Thus, polarization-resolved non-linear optical microscopy provides high image contrast and quantitative estimate of sample orientation. An overview of the advancements on polarization-resolved SHG microscopy including Stokes vector based polarimetry, circular dichroism, and susceptibility are also presented in this review article. The working principles and corresponding implements of above-mentioned microscopy techniques are elucidated. The potential of time-resolved TPF lifetime imaging microscopy (TP-FLIM) is explored by imaging endogenous fluorescence of NAD(P)H, a key coenzyme in cellular metabolic processes. We also discuss single laser source time-resolved multimodal CARS-FLIM microscopy using time-correlated single-photon counting (TCSPC) in combination with continuum generation from photonic crystal fiber (PCF). Using examples, we demonstrate that the multimodal NLO microscopy is a powerful tool to assess the molecular specificity with high resolution.