Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods.

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In eukaryotic cells, chromatin structure is linked to transcription processes through the regulation of genome organization. Extending across multiple length-scales -from the nucleosome to higher-order three-dimensional structures -chromatin is a dynamic system which evolves throughout the lifetime of a cell. However, no individual technique can fully elucidate the behavior of chromatin organization and its relation to molecular function at all length-and time-scales at both a single-cell and a cell population level. Herein, we present a multi-technique nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform that bridges electron tomography and optical superresolution imaging of chromatin conformation and transcriptional processes, with resolution down to the level of individual nucleosomes, with high-throughput, label-free analysis of chromatin packing and its dynamics in live cells. Utilizing nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nm, sub-Mb genomic size, and an internal fractal structure. The chromatin packing behavior of these domains is directly influenced by active gene transcription. Furthermore, we demonstrated that the chromatin packing domain structure is correlated among progenitor cells and all their progeny, indicating that the organization of chromatin into fractal packing domains is heritable across cell division. Further studies employing the nano-ChIA platform have the potential to provide a more coherent picture of chromatin structure and its relation to molecular function.

Purpose: We aimed to analyze whether ERG rearrangement in biopsies could be used to assess subsequent cancer diagnosis in high-grade prostatic intraepithelial neoplasia (HGPIN) and the risk of lymph node metastasis in early prostate cancer.Experimental Design: Samples from 523 patients (361 with early prostate cancer and 162 with HGPIN) were collected prospectively. On the basis of the cutoff value established previously, the 162 patients with HGPIN were stratified to two groups: one with an ERG rearrangements rate !1.6% (n ¼ 59) and the other with an ERG rearrangements rate <1.6% (n ¼ 103). For the 361 prostate cancer cases undergoing radical prostatectomy, 143 had pelvic lymph node dissection (node-positive, n ¼ 56 and node-negative, n ¼ 87). All ERG rearrangement FISH data were validated with ERG immunohistochemistry.Results: A total of 56 (of 59, 94.9%) HGPIN cases with an ERG rearrangements rate !1.6% and 5 (of 103, 4.9%) HGPIN cases with an ERG rearrangements rate <1.6% were diagnosed with prostate cancer during repeat biopsy follow-ups (P < 0.001). There were significant differences in ERG rearrangement rates between lymph node-positive and -negative prostate cancer (P < 0.001). The optimal cutoff value to predict lymph node metastasis by ERG rearrangement was established, being 2.6% with a sensitivity at 80.4% [95% confidence interval (CI), 67.6-89.8] and a specificity at 85.1% (95% CI, 75.8-91.8). ERG protein expression by immunohistochemistry was highly concordant with ERG rearrangement by FISH.Conclusions: The presence of ERG rearrangement in HGPIN lesions detected on initial biopsy warrants repeat biopsies and measuring ERG rearrangement could be used for assessing the risk of lymph node metastasis in early prostate cancer. Clin Cancer Res; 18(15); 4163-72. Ó2012 AACR.