Scott Gladstein

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

Significance Chromatin is one of the most critical structures within the cell because it houses most genetic information. Its structure is well understood at the nucleosomal (<20-nm) and chromosomal (>200-nm) 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 and 200 nm that can quantify the nanoarchitecture 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 study of the relationship between nanoscale organization and molecular function. 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.

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.

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.A model accounting for the properties of the local chromatin environment predicts the modulation of patterns in gene expression and helps screen for chemotherapeutic adjuvants that lead to an enhanced therapeutic response in cancer cells.

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

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 Statement Chromatin 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.

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 technologys promise as an early cancer screening technology.

Multimodal interferometric imaging of nanoscale structure and macromolecular motion uncovers UV induced cellular paroxysm

We present a multimodal label-free interferometric imaging platform for measuring intracellular nanoscale structure and macromolecular dynamics in living cells with a sensitivity to macromolecules as small as 20nm and millisecond temporal resolution. We validate this system by pairing experimental measurements of nanosphere phantoms with a novel interferometric theory. Applying this system in vitro, we explore changes in higher-order chromatin structure and dynamics that occur due to cellular fixation, stem cell differentiation, and ultraviolet (UV) light irradiation. Finally, we discover a new phenomenon, cellular paroxysm, a near-instantaneous, synchronous burst of motion that occurs early in the process of UV induced cell death. Given this platform’s ability to obtain nanoscale sensitive, millisecond resolved information within live cells without concerns of photobleaching, it has the potential to answer a broad range of critical biological questions about macromolecular behavior in live cells, particularly about the relationship between cellular structure and function.

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.

Multimodal nanoscale imaging of chromatin with super resolution microscopy and partial wave spectroscopy (Conference Presentation)

We demonstrate a multimodal imaging methodology to probe the nanoscale environment of cells. The system combines partial-wave spectroscopic (PWS) microscopy and spectroscopic photon localization microscopy (SPLM). PWS quantifies the nanoarchitecture of cells with sensitivity to structures between 20 and 200 nm. SPLM is a newly developed super-resolution imaging technique based upon the principles of single-molecule localization microscopy and spectroscopy. In addition to allowing super-resolution imaging, SPLM provides inherent molecular-specific spectroscopic information of targeted structures visualized when dyes are used. Combining both of these modalities into a single instrument allows nanoscale characterization of the super-resolution molecular imaging provided by SPLM as it relates to nanoscale structural information provided by PWS. As an example, we labeled RNA polymerase in HeLa cells and showed correlations between the locations of the RNA polymerase visualized by SPLM and the nanoscale structure of the chromatin measured by PWS. Such information is crucial in understanding the role of specific molecules in regulating the chromatin structure and gene expression. More broadly, this instrument can give insight into the molecular pathways of diseases and therapeutic treatments of those diseases, while simultaneously showing the effects on chromatin topology.

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 patients 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.

Abstract A25: A novel spectroscopic technology to image the native chromatin nanostructure in live cells

Proper regulation of higher-order chromatin structure is essential for normal gene regulation and cellular function. We have previously found that the nanoscale chromatin structure is significantly altered in early and field carcinogenesis using novel spectroscopic methods in parallel with biological assays (Backman and Roy, J Cancer, 2013, 3:251-261; Subramanian et al, Cancer Res, 2009, 13:5357-63; Stypula-Cyrus et al, PLoS One, 2013, 5:e64600). This was done in fixed human and animal model samples, suggesting that genetic/epigenetic alterations can serve as the earliest marker for neoplastic transformation. While chromatin is well understood at the nucleosomal level ( 200nm), little is known about the higher-order chromatin structure between these length scales. Current techniques available to study cellular structures below the diffraction limit ( The BaSIS instrument was built into a commercial inverted microscope (Leica DMIRB) equipped with a high NA oil immersion objective with broadband illumination provided by a Xenon lamp. Refractive index fluctuations are measured by sampling backscattered light at each wavelength 500-700nm using a combination of a liquid crystal tunable filter (LCTF) and a CMOS camera. HeLa and CHO cells were first imaged in petri dishes with coverslip bottoms, and then incubated with Hoechst 33342, a nuclear stain that binds to AT-rich regions of the genome and has been reported to cause double-stranded breaks (DSBs) in the DNA (Pfeiffer et al, Mutagenesis, 2000, 4: 289-302). Additionally, mock-staining experiments were performed to compare the changes in nuclear structure due to Hoechst 33342 excitation compared to UV light exposure alone. We utilized a γ-H2A.X-Alexa488 conjugated antibody after Hoechst- and mock-staining to compare observed changes in BaSIS signal with the formation of DSBs. Using BaSIS, we show for the first time that the excitation of Hoechst 33342 immediately alters the native nuclear nanostructure and induces formation of DSBs, confirmed by the rapid phosphorylation of H2A.X. In our mock-stained control, we observed an average increase of 0.006% and 0.001% signal after UV exposure (p-value > 0.5), whereas the stained cells display a 17.01% and 7.1% decrease in HeLa and CHO nuclei, respectively (p-value In conclusion, BaSIS is a powerful tool for studying the dynamics of chromatin nanostructure and can serve as a natural supplement to super-resolution fluorescence techniques, providing quantified information about native cellular organization. With this technique, we demonstrated that using the Hoechst DNA-binding dye causes irreversible alterations in chromatin structure at time-scales (seconds) not previously recognized. As a result, BASIS can be applied to a broad range of critical studies in chromatin research. Current and future research include: (i) mRNA transport and the accessibility of euchromatin and heterochromatin to transcription factors; (ii) why and how high-order chromatin structure changes in cancer progression; (iii) the role of nuclear architecture as an epigenetic regulator of gene expression; and (iv) the effect of metabolism on chromatin structure; (v) damage/repair mechanisms and potentially, chemotherapeutic efficacy. Citation Format: Yolanda Stypula, Scott Gladstein, Luay Almassalha, Greta Bauer, John Chandler, Lusik Cherkezyan, Di Zhang, Hariharan Subramanian, Igal Szleifer, Vadim Backman. A novel spectroscopic technology to image the native chromatin nanostructure in live cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A25.