Live Cell Partial Wave Spectroscopic microscopy: Label-free Imaging of the Native, Living Cellular Nano-architecture

Abstract

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10nm) to the chromosomal (>200nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using Partial Wave Spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20-200nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nano-architecture. Therefore, we developed a live cell PWS technique which allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real-time. In this work, we employ live cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live cell DNA binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Since biological function is tightly paired with structure, live cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.Significance StatementChromatin is one of the most critical structures within the cell because it houses most genetic information. Its structure is well understood at the nucleosomal (<20nm) and chromosomal (>200nm) levels, however, due to the lack of quantitative imaging modalities to study this organization, little is known about the higher-order structure between these length scales in live cells. We present a label-free technique, live cell Partial Wave Spectroscopic (PWS) microscopy with sensitivity to structures between 20-200nm that can quantify the nano-architecture in live cells. With this technique, we can detect DNA fragmentation and expand on the link between metabolic function and higher-order chromatin structure. Live cell PWS allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in live cells.

Macrogenomic engineering via modulation of the scaling of chromatin packing density

“…Live-cell PWS microscopy measurements were performed on HT-29 cells grown on 5 mm glass bottom petri dishes (Cell Vis) and serum starved for 5 hours before and after stimulation 59 . Changes in gene expression for each treatment group were measured using Illumina human HG12-T microarray chips of mRNA collected by TRIzol isolation (Life Technologies, Carlsbad California) from 10 mL petri dishes.…”

The Global Relationship between Chromatin Physical Topology, Fractal Structure, and Gene Expression

“…The resulting relative L D for each group was 0.9, 1.0, 0.98, 0.8, 0.64, and 0.68 for CV SD, CV SE, EGF, PMA, A SD, and A SE, respectively. Live cell PWS measurements were performed on HT-29 cells grown on 5 mm glass bottom petri dishes (Cell Vis) and serum starved for 5 hours63. Cells were maintained at 37 C and 5% CO 2 for the duration of the experiment.…”

Review of interferometric spectroscopy of scattered light for the quantification of subdiffractional structure of biomaterials

“…In addition, we discussed the different approaches to data analysis which can be taken depending on the characteristics of studied samples, such as degree of nanoscale surface roughness and sample thicknesses, as well as on how important the spatial resolution of nanoscale information (Σðx; yÞ) and the speed of data acquisition (Σ p ) are for the particular application. Similarly, some of the instrumentation features can be adjusted to achieve the desired depth of focus, or the sample geometry can be altered to allow measurements from other sample types, such as living biological cells, 18,49 while satisfying the requirements of the technique. From the instrumentation perspective, the SM technique is quite simple and can even be built into a commercial microscope.…”