Label-free imaging of cell nuclear dynamics by coherence brightfield (COBRI) microscopy

Yi-Teng Hsiao^1*, Chia-Ni Tsai¹, Chia-Lung Hsieh¹

¹Institute of Atomic and Molecular Sciences, Academia Sinica, Taiwan

* Presenter:Yi-Teng Hsiao, email:tonyhsian@gmail.com

Optical microscope imaging is a unique tool to measure biological structures and dynamics at high spatiotemporal resolutions. Current bioimaging has relied on fluorescence labeling because of its superb sensitivity and its high specificity. However, it has encountered several challenges and fundamental limitations, such as photobleaching, blinking, and saturation. Immunostaining requires sample fixation, which interrupts continuous observations of cell dynamics. Recent advance in scattering-based imaging has circumvented the fundamental problems of fluorescence and thus become a powerful imaging modality. For example, by detecting linear scattering light through interference, very small single molecules can be detected without labeling. Despite the great success of scattering-based imaging, previous highly sensitive measurements were performed on purified samples and in a well-controlled environment of a clean cover glass. It remains unclear how scattering-based imaging could be operated for live cells where the sample is densely packed with a great diversity of molecules. Moreover, unlike fluorescence labeling, label-free linear scattering signal does not have specificity (every biomolecule scatters light!), which greatly complicates the data interpretation.

Here, using cell nucleus as an example, we investigate how to perform highly sensitive scattering-based imaging and extract biology-relevant information. The cell nucleus is densely packed with DNA and proteins. In particular, the DNA is wrapped on histone proteins, which together are known as chromatin. We use Coherent Brightfield microscopy (COBRI) developed in our lab in the past few years to measure the intrinsic scattering signal of chromatin through interference. The COBRI video recorded at a high speed (>1k Hz) for a few seconds, which captures the dynamic scattering signal due to the spatially fluctuating chromatin. We establish a model to interpret the dynamic COBRI signal from which the local dynamic properties of chromatin can be estimated (including the size, volume fraction, and diffusion constant). The model is validated by measuring the diffusion properties of nanoparticle colloids. Using our label-free methods, we are able to map the local chromatin dynamics at high spatiotemporal resolutions. In addition, since our COBRI imaging does not suffer photobleaching, we immediately succeeded in long-term and high-speed chromatin imaging. Our data shows distinct dynamics between nucleoli and nucleoplasm. Within the nucleoplasm regions, we are able to distinguish condensation states of chromatin, i.e., heterochromatin and euchromatin. This work demonstrates the potential of COBRI imaging to reveal cell structures and dynamics with minimal perturbations and thus shows promise of exploring new cell biology.

Keywords: label-free microscopy, scattering-based imaging, cell nuclear dynamics, chromatin condensation