中央研究院 原子與分子科學研究所

Chromatin is a DNA–protein complex that is densely packed in the cell nucleus. The nanoscale chromatin compaction plays critical roles in the modulation of cell nuclear processes. However, little is known about the spatiotemporal dynamics of chromatin compaction states because it remains difficult to quantitatively measure the chromatin compaction level in live cells. Here, we demonstrate a strategy, referenced as DYNAMICS imaging, for mapping chromatin organization in live cell nuclei by analyzing the dynamic scattering signal of molecular fluctuations. Highly sensitive optical interference microscopy, coherent brightfield (COBRI) microscopy, is implemented to detect the linear scattering of unlabeled chromatin at a high speed. A theoretical model is established to determine the local chromatin density from the statistical fluctuation of the measured scattering signal. DYNAMICS imaging allows us to reconstruct a speckle-free nucleus map that is highly correlated to the fluorescence chromatin image. Moreover, together with calibration based on nanoparticle colloids, we show that the DYNAMICS signal is sensitive to the chromatin compaction level at the nanoscale. We confirm the effectiveness of DYNAMICS imaging in detecting the condensation and decondensation of chromatin induced by chemical drug treatments. Importantly, the stable scattering signal supports a continuous observation of the chromatin condensation and decondensation processes for more than 1 h. Using this technique, we detect transient and nanoscopic chromatin condensation events occurring on a time scale of a few seconds. Label-free DYNAMICS imaging offers the opportunity to investigate chromatin conformational dynamics and to explore their significance in various gene activities.

Link to the paper: https://pubs.acs.org/doi/10.1021/acsnano.1c09748
Recommended in Faculty Opinion: https://facultyopinions.com/prime/741388510
Reported by Science Promotion & Engagement Center: https://spec.ntu.edu.tw/20220322-research-chem/
 

Light absorption is a common phenomenon in nature, but accurate and quantitative absorption measurement at the nanoscale remains challenging especially in the application of widefield imaging. Here, we demonstrated optical widefield interferometric photothermal microscopy that allowed us to visualize and quantify the heat generation of single nanoparticles. The working principle was to measure the scattering signal due to the refractive index change of the surrounding media induced by the dissipated heat (known as the thermal lens effect). The sensitivity of our local heat measurement was a few nanowatts—the high sensitivity made it possible to detect single gold nanoparticles, as small as 5 nm. By changing the particle sizes, we found that, for small metallic nanoparticles (gold and silver nanoparticles < 40 nm), the photothermal signal was determined by the amount of the dissipated heat, independent of the particle size. A model was established to explain our experimental results, indicating that the photothermal signal was essentially contributed by the interferometric detection of the scattered field of the thermal lens. Importantly, on the basis of this model, we further investigated the photothermal signal of large nanoparticles (40–100 nm for our setup) where the scattered light of the particle was considerable relative to the probe light. In this regime, the strong scattered field of the particle effectively served as the main reference beam that interfered with the scattered field of the thermal lens, resulting in an enhanced photothermal signal. Our work illustrates an important fact that the measured photothermal signal is fundamentally affected by the scattering property of the sample. This finding paves the way to accurate and sensitive absorption-based imaging in complex biological samples where the scattering is often spatially heterogeneous.
 

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 cross-linking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects using nanoparticles to measure the diffusion of single-membrane molecules. We 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 shows fast and free diffusion in supported lipid bilayers (2–3 μm²/s for rAv–AuNP sizes of 10–40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and cross-linking effects are successfully avoided. Our work shows that the measured diffusion of the membrane molecule is highly sensitive to the molecular design of the cross-linker 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.

This work is selected as 2019 Significant Research Achievement of Academia Sinica and reported by ACS Nano Perspective (Yanqi Yu, Miao Li, Yan Yu. Tracking Single Molecules in Biomembranes: Is Seeing Always Believing?. ACS Nano 2019, 13 (10) , 10860-10868.)

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.

Localization of single nano-sized 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, 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 sub-diffraction limit 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 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.

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.