Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy.

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 (10-nm) to the chromosomal (>200-nm) 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 and 200 nm, 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 nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use 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. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.

Correction: Label free localization of nanoparticles in live cancer cells using spectroscopic microscopy.

Correction for 'Label free localization of nanoparticles in live cancer cells using spectroscopic microscopy' by Graham L. C. Spicer et al., Nanoscale, 2018, 10, 19125-19130, https://doi.org/10.1039/C8NR07481J.

Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function.

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.

Preservation of cellular nano-architecture by the process of chemical fixation for nanopathology.

Transformation in chromatin organization is one of the most universal markers of carcinogenesis. Microscale chromatin alterations have been a staple of histopathological diagnosis of neoplasia, and nanoscale alterations have emerged as a promising marker for cancer prognostication and the detection of predysplastic changes. While numerous methods have been developed to detect these alterations, most methods for sample preparation remain largely validated via conventional microscopy and have not been examined with nanoscale sensitive imaging techniques. For these nanoscale sensitive techniques to become standard of care screening tools, new histological protocols must be developed that preserve nanoscale information. Partial Wave Spectroscopic (PWS) microscopy has recently emerged as a novel imaging technique sensitive to length scales ranging between 20 and 200 nanometers. As a label-free, high-throughput, and non-invasive imaging technique, PWS microscopy is an ideal tool to quantify structural information during sample preparation. Therefore, in this work we applied PWS microscopy to systematically evaluate the effects of cytological preparation on the nanoscales changes of chromatin using two live cell models: a drug-based model of Hela cells differentially treated with daunorubicin and a cell line comparison model of two cells lines with inherently distinct chromatin organizations. Notably, we show that existing cytological preparation can be modified in order to maintain clinically relevant nanoscopic differences, paving the way for the emerging field of nanopathology.

Multimodal interference-based imaging of nanoscale structure and macromolecular motion uncovers UV induced cellular paroxysm.

Understanding the relationship between intracellular motion and macromolecular structure remains a challenge in biology. Macromolecular structures are assembled from numerous molecules, some of which cannot be labeled. Most techniques to study motion require potentially cytotoxic dyes or transfection, which can alter cellular behavior and are susceptible to photobleaching. Here we present a multimodal label-free imaging platform for measuring intracellular structure and macromolecular dynamics in living cells with a sensitivity to macromolecular structure as small as 20 nm and millisecond temporal resolution. We develop and validate a theory for temporal measurements of light interference. In vitro, we study how higher-order chromatin structure and dynamics change during cell differentiation and ultraviolet (UV) light irradiation. Finally, we discover cellular paroxysms, a near-instantaneous burst of macromolecular motion that occurs during UV induced cell death. With nanoscale sensitive, millisecond resolved capabilities, this platform could address critical questions about macromolecular behavior in live cells.

Measuring Nanoscale Chromatin Heterogeneity with Partial Wave Spectroscopic Microscopy.

Despite extensive research in the area, current understanding of the structural organization of higher-order chromatin topology (between 20 and 200 nm) is limited due to a lack of proper imaging techniques at these length scales. The organization of chromatin at these scales defines the physical context (nanoenvironment) in which many important biological processes occur. Improving our understanding of the nanoenvironment is crucial because it has been shown to play a critical functional role in the regulation of chemical reactions. Recent progress in partial wave spectroscopic (PWS) microscopy enables real-time measurement of higher-order chromatin organization within label-free live cells. Specifically, PWS quantifies the nanoscale variations in mass density (heterogeneity) within the cell. These advancements have made it possible to study the functional role of chromatin topology, such as its regulation of the global transcriptional state of the cell and its role in the development of cancer. In this chapter, the importance of studying chromatin topology is explained, the theory and instrumentation of PWS are described, the measurements and analysis processes for PWS are laid out in detail, and common issues, troubleshooting steps, and validation techniques are provided.

Correlating colorectal cancer risk with field carcinogenesis progression using partial wave spectroscopic microscopy.

Prior to the development of a localized cancerous tumor, diffuse molecular, and structural alterations occur throughout an organ due to genetic, environmental, and lifestyle factors. This process is known as field carcinogenesis. In this study, we used partial wave spectroscopic (PWS) microscopy to explore the progression of field carcinogenesis by measuring samples collected from 190 patients with a range of colonic history (no history, low-risk history, and high-risk history) and current colon health (healthy, nondiminutive adenomas (NDA; ≥5 mm and <10 mm), and advanced adenoma [AA; ≥10 mm, HGD, or >25% villous features]). The low-risk history groups include patients with a history of NDA. The high-risk history groups include patients with either a history of AA or colorectal cancer (CRC). PWS is a nanoscale-sensitive imaging technique which measures the organization of intracellular structure. Previous studies have shown that PWS is sensitive to changes in the higher-order (20-200 nm) chromatin topology that occur due to field carcinogenesis within histologically normal cells. The results of this study show that these nanoscale structural alterations are correlated with a patient's colonic history, which suggests that PWS can detect altered field carcinogenic signatures even in patients with negative colonoscopies. Furthermore, we developed a model to calculate the 5-year risk of developing CRC for each patient group. We found that our data fit this model remarkably well (R2  = 0.946). This correlation suggests that PWS could potentially be used to monitor CRC progression less invasively and in patients without adenomas, which opens PWS to many potential cancer care applications.

Label free localization of nanoparticles in live cancer cells using spectroscopic microscopy.

Gold nanoparticles (GNPs) have become essential tools used in nanobiotechnology due to their tunable plasmonic properties and low toxicity in biological samples. Among the available approaches for imaging GNPs internalized by cells, hyperspectral techniques stand out due to their ability to simultaneously image and perform spectral analysis of GNPs. Here, we present a study utilizing a recently introduced hyperspectral imaging technique, live-cell PWS, for the imaging, tracking, and spectral analysis of GNPs in live cancer cells. Using principal components analysis, the extracellular or intracellular localization of the GNPs can be determined without the use of exogenous labels. This technique uses wide-field white light, assuring minimal toxicity and suitable signal-to-noise ratio for spectral and temporal resolution of backscattered signal from GNPs and local cellular structures. The application of live-cell PWS introduced here could make a great impact in nanomedicine and nanotechnology by giving new insights into GNP internalization and intracellular trafficking.

The transformation of the nuclear nanoarchitecture in human field carcinogenesis.

Morphological alterations of the nuclear texture are a hallmark of carcinogenesis. At later stages of disease, these changes are well characterized and detectable by light microscopy. Evidence suggests that similar albeit nanoscopic alterations develop at the predysplastic stages of carcinogenesis. Using the novel optical technique partial wave spectroscopic microscopy, we identified profound changes in the nanoscale chromatin topology in microscopically normal tissue as a common event in the field carcinogenesis of many cancers. In particular, higher-order chromatin structure at supranucleosomal length scales (20-200 nm) becomes exceedingly heterogeneous, a measure we quantify using the disorder strength (Ld ) of the spatial arrangement of chromatin density. Here, we review partial wave spectroscopic nanocytology clinical studies and the technology's promise as an early cancer screening technology.

Macrogenomic engineering via modulation of the scaling of chromatin packing density.

Many human diseases result from the dysregulation of the complex interactions between tens to thousands of genes. However, approaches for the transcriptional modulation of many genes simultaneously in a predictive manner are lacking. Here, through the combination of simulations, systems modelling and in vitro experiments, we provide a physical regulatory framework based on chromatin packing-density heterogeneity for modulating the genomic information space. Because transcriptional interactions are essentially chemical reactions, they depend largely on the local physical nanoenvironment. We show that the regulation of the chromatin nanoenvironment allows for the predictable modulation of global patterns in gene expression. In particular, we show that the rational modulation of chromatin density fluctuations can lead to a decrease in global transcriptional activity and intercellular transcriptional heterogeneity in cancer cells during chemotherapeutic responses to achieve near-complete cancer cell killing in vitro. Our findings represent a 'macrogenomic engineering' approach to modulating the physical structure of chromatin for whole-scale transcriptional modulation.

Role of Mitochondrial Oxidative Stress in Glucose Tolerance, Insulin Resistance, and Cardiac Diastolic Dysfunction.

BACKGROUND: Diabetes mellitus (DM) is associated with mitochondrial oxidative stress. We have shown that myocardial oxidative stress leads to diastolic dysfunction in a hypertensive mouse model. Therefore, we hypothesized that diabetes mellitus could cause diastolic dysfunction through mitochondrial oxidative stress and that a mitochondria-targeted antioxidant (MitoTEMPO) could prevent diastolic dysfunction in a diabetic mouse model. METHODS AND RESULTS: C57BL/6J mice were fed either 60 kcal % fat diet (high-fat diet [HFD]) or normal chow (control) for 8 weeks with or without concurrent MitoTEMPO administration, followed by in vivo assessment of diastolic function and ex vivo studies. HFD mice developed impaired glucose tolerance compared with the control (serum glucose=495±45 mg/dL versus 236±30 mg/dL at 60 minutes after intraperitoneal glucose injection, P<0.05). Myocardial tagged cardiac magnetic resonance imaging showed significantly reduced diastolic circumferential strain (Ecc) rate in the HFD mice compared with controls (5.0±0.3 1/s versus 7.4±0.5 1/s, P<0.05), indicating diastolic dysfunction in the HFD mice. Systolic function was comparable in both groups (left ventricular ejection fraction=66.4±1.4% versus 66.7±1.2%, P>0.05). MitoTEMPO-treated HFD mice showed significant reduction in mitochondria reactive oxygen species, S-glutathionylation of cardiac myosin binding protein C, and diastolic dysfunction, comparable to the control. The fasting insulin levels of MitoTEMPO-treated HFD mice were also comparable to the controls (P>0.05). CONCLUSIONS: MitoTEMPO treatment prevented insulin resistance and diastolic dysfunction, suggesting that mitochondrial oxidative stress may be involved in the pathophysiology of both conditions.

The Greater Genomic Landscape: The Heterogeneous Evolution of Cancer.

Results have historically shown a broad plasticity in the origin of tumors and their functions, with significant heterogeneity observed in both morphologies and functional capabilities. Largely unknown, however, are the mechanisms by which these variations occur and how these events influence tumor formation and behavior. Contemporary views on the origin of tumors focus mainly on the role of particular sets of driver transformations, mutational or epigenetic, with the occurrence of the observed heterogeneity as an accidental byproduct of oncogenesis. As such, we present a hypothesis that tumors form due to heterogeneous adaptive selection in response to environmental stress through intrinsic genomic sampling mechanisms. Specifically, we propose that eukaryotic cells intrinsically explore their available genomic information, the greater genomic landscape (GGL), in response to stress under normal conditions, long before the formation of a cancerous lesion. Finally, considering the influence of chromatin heterogeneity on the GGL, we propose a new class of compounds, chromatin-protective therapies (CPT), which target the physical variations in chromatin topology. In this approach, CPTs reduce the overall information space available to limit the formation of tumors or the development of drug-resistant phenotypes. Cancer Res; 76(19); 5605-9. ©2016 AACR.