Nuclear morphology measurements with angle-resolved low coherence interferometry for application to cell biology and early cancer detection

The study of intact, living cells using non-invasive optical spectroscopic methods offers the opportunity to assess cellular structure and organization in a way that is not possible with commonly used cell biology imaging techniques. We have developed a novel spectroscopic technique for diagnosing disease at the cellular level based on using low-coherence interferometry (LCI) to detect the angular distribution of scattered light. Angle-resolved LCI (a/LCI) combines the ability of LCI to isolate scattering from sub-surface tissue layers with the ability of light scattering spectroscopy to obtain structural information on sub-wavelength scales. In application to examining cellular structure, a/LCI enables quantitative measurements of changes in the size and texture of cell nuclei. These quantitative measurements are characteristic of different pathological states. The capabilities of a/LCI were demonstrated using a clinical system that can be applied in endoscopic surveillance of esophageal tissue, producing high sensitivity and specificity for detecting dysplastic tissues in vivo. Experiments with in vitro cell samples also show the utility of a/LCI in observing structural changes due to environmental stimuli as well as detecting apoptosis due to chemotherapeutic agents.     

Angular range,sampling and noise considerations for inverse light scattering analysis of nuclear morphology

In recent years, significant work has been devoted to the use of angle‐resolved elastic scattering for the extraction of nuclear morphology in tissue. By treating the nucleus as a Mie scattering object, techniques such as angle‐resolved low‐coherence interferometry (a/LCI) have demonstrated substantial success in identifying nuclear alterations associated with dysplasia. Because optical biopsies are inherently noninvasive, only a small, discretized portion of the 4π scattering field can be collected from tissue, limiting the amount of information available for diagnostic purposes. In this work, we comprehensively characterize the diagnostic impact of variations in angular sampling, range and noise for inverse light scattering analysis of nuclear morphology, using a previously reported dataset from 40 patients undergoing a/LCI optical biopsy for cervical dysplasia. The results from this analysis are applied to a benchtop scanning a/LCI system which compromises angular range for wide‐area scanning capability. This work will inform the design of next‐generation optical biopsy probes by directing optical design towards parameters which offer the most diagnostic utility.      

Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei

The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.     

Measuring topology of low-intensity DNA methylation sites for high-throughput assessment of epigenetic drug-induced effects in cancer cells

Epigenetic anti-cancer drugs with demethylating effects have shown to alter genome organization in mammalian cell nuclei. The interest in the development of novel epigenetic drugs has increased the demand for cell-based assays to evaluate drug performance in pre-clinical studies. An imaging-based cytometrical approach that can measure demethylation effects as changes in the spatial nuclear distributions of methylated cytosine and global DNA in cancer cells is introduced in this paper. The cells were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei. In the preprocessing step the segmentation of nuclei in three-dimensional images (3-D) is followed by an automated assessment of nuclear DAPI/MeC patterns to exclude dissimilar entities. Next, low-intensity MeC (LIM) and low-intensity DNA (LID) sites of similar nuclei are localized and processed to obtain specific nuclear density profiles. These profiles sampled at half of the total nuclear volume yielded two parameters: LIM_0.5 and LID_0.5. The analysis shows that zebularine and 5-azacytidine—the two tested epigenetic drugs introduce changes in the spatial distribution of low-intensity DNA and MeC signals. LIM_0.5 and LID_0.5 were significantly different (p < 0.001) in 5-azacytidine treated (n = 660) and zebularine treated (n = 496) vs. untreated (n = 649) DU145 human prostate cancer cells. In the latter case the LIM sites were predominantly found at the nuclear border, whereas treated populations showed different degrees of increase in LIMs towards the interior nuclear space, in which a large portion of heterochromatin is located. The cell-by-cell evaluation of changes in the spatial reorganization of MeC/DAPI signals revealed that zebularine is a more gentle demethylating agent than 5-azacytidine. Measuring changes in the topology of low-intensity sites can potentially be a valuable component in the high-throughput assessment of demethylation and risk of chromatin reorganization in epigenetic-drug screening tasks.     

A Nondimensional Model Reveals Alterations in Nuclear Mechanics upon Hepatitis C Virus Replication

Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of β-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.     

Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains

We report a new approach to probing DNA-protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin-streptavidin linkage withstands ∼20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ?65 pN; access to this higher force regime is sufficient to probe the responses of protein-DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.     

Probing compressibility of the nuclear interior in wild-type and lamin deficient cells using microscopic imaging and computational modeling

Mechanical properties of the cell nucleus play an important role in maintaining the integrity of the genome and controlling the cellular force balance. Irregularities in these properties have been related to disruption of a variety of force-dependent processes in the cell, such as migration, division, growth or differentiation. Characterizing mechanical properties of the cell nucleus in situ and relating these parameters to cellular phenotypes remain challenging tasks, as conventional micromanipulation techniques do not allow direct probing of intracellular structures. Here, we present a framework based on light microscopic imaging and automated mechanical modeling that enables characterization of the compressibility of the nuclear interior in situ. Based entirely on optical methods, our approach does not require application of destructive or contacting techniques and it enables measurements of a significantly larger number of cells. Compressibility, in this paper represented by Poisson's ratio ν, is determined by fitting a numerical model to experimentally observed time series of microscopic images of fluorescent cell nuclei in which bleached patterns are introduced. In a proof-of-principle study, this framework was applied to estimate ν in wild type cells and cells lacking important structural proteins of the nuclear envelope (LMNA(-/-)). Based on measurements of a large number of cells, our study revealed distinctive changes in compressibility of the nuclear interior between these two cell types. Our method allows an automated, contact-free estimation of mechanical properties of intracellular structures. Combined with knockdown and overexpression screens, it paves the way towards a high-throughput measurement of intracellular mechanical properties in functional phenotyping screens.     

Age-associated reduction of nuclear shape dynamics in excitatory neurons of the visual cortex

Neurons decline in their functionality over time, and age-related neuronal alterations are associated with phenotypes of neurodegenerative diseases. In nonneural tissues, an infolded nuclear shape has been proposed as a hallmark of aged cells and neurons with infolded nuclei have also been reported to be associated with neuronal activity. Here, we performed time-lapse imaging in the visual cortex of Nex-Cre;SUN1-GFP mice. Nuclear infolding was observed within 10 min of stimulation in young nuclei, while the aged nuclei were already infolded pre-stimulation and showed reduced dynamics of the morphology. In young nuclei, the depletion of the stimuli restored the nucleus to a spherical shape and reduced the dynamic behavior, suggesting that nuclear infolding is a reversible process. We also found the aged nucleus to be stiffer than the young one, further relating to the age-associated loss of nuclear shape dynamics. We reveal temporal changes in the nuclear shape upon external stimulation and observe that these morphological dynamics decrease with age.     

High-throughput measuring of meiotic recombination rates in barley pollen nuclei using Crystal Digital PCRTM

Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole-genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high-throughput measurements of meiotic recombination rates in barley pollen nuclei without whole-genome amplification were performed through a Crystal Digital PCR^TM-based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42 000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCR^TM enables reliable, rapid and high-throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei.     

Polyanion induced physical changes in isolated nuclei

Small angle light scattering techniques were used to determine the effects of selected polyanions on isolated mouse spleen nuclei. The light scattering measurements made it possible to distinguish nuclear swelling from lysis and/or aggregation. Heparin and dextran sulfate were shown to induce nuclear swelling. Chondroitin sulfate, although structurally similar to heparin, had no observable effect on the nuclei. Polystyrene sulfonate and pyran copolymer caused nuclear aggregation and lysis, respectively.     

Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight X 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heat-stressed nuclei showed a relationship between dosage and the coefficient of variation of alpha t [red/(red + green fluorescence)] measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single-stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as alpha t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.     

Wavelength-Ratiometric Plasmon Light Scattering-Based Immunoassays

The application of a wavelength-ratiometric plasmon light scattering technique to immunoassays is demonstrated. A model immunoassay for anti-immunoglobulin G (IgG), constructed in gold colloid-modified high-throughput screening wells, was monitored by the changes in the intensity of scattered light (with transmitted light) from gold colloids as a result of antibody–antibody interactions. The quantitative determination of anti-IgG was undertaken by measuring the ratio of intensity of scattered light at both 590 and 500 nm. A white light-emitting diode (LED) and a fiber optic coupled fluorometer was used as an excitation source and the detection system, respectively. The visual confirmation of the quantitative nature of the measurement technique was done by digital photography. A lower detection limit of 0.05 μg/mL for anti-IgG was determined. The wavelength-ratiometric plasmon light scattering technique offers several advantages: (1) light at >500 nm can be used for reduced biological autofluorescence; (2) due to the ratiometric nature of these measurements, the fluctuations in the excitation or ambient light do not perturb the measured signal; and (3) with the addition of automated detection systems, multiple samples in a high-throughput format can potentially be assessed quickly and more efficiently.     

Follicle and oocyte morphology in ewes after treatment with insulin in the late follicular phase

Stress reduces fertility in ruminants. Various experimental models, such as insulin-induced hypoglycaemia, have been used to investigate the mechanisms involved, and have revealed abnormal LH profiles (both pulse and surge secretion). This disruption affects follicular function and it is proposed there may be negative consequences on subsequent oocyte morphology. Insulin (5 iu/kg), administered to ewes in the late follicular phase, induced hypoglycemia for 10 h, decreased estradiol concentrations for 8-12 h and delayed the LH surge by 15 h. Although the diameters of dominant follicles just before ovulation were not affected, granulosa cells had fewer pycnotic nuclei, less apoptosis and increased proliferation 16-17 h after the LH surge. Nevertheless, we did not observe gross ultra-structural differences in nuclear, cytoplasmic or cumulus maturity between oocytes from insulin-treated and control animals. This suggests that reduced LH pulsatility and a delay in the LH surge may only produce very subtle changes in gross oocyte morphology, imperceptible by electron microscopy.     

THE ISOLATION OF CELL NUCLEI FROM RAT BRAIN

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.     

Alterations in nuclear anatomy by chemical modification of proteins in isolated rat liver nuclei

Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, approximately 2-4s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while approx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA [less than or equal to 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli, and ribonucleoprotein (predominantly hnRNA group A and B). These nucleic acid-deficient structures have been termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants, or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA treatment), but missing the double bilayer and a 51K polypeptide that is a major component of the other structures. The use of chemical modification on the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve two functions: (a) as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; (b) offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.     

LCI data and data quality

Life cycle inventory (LCI) is becoming an established environmental management tool that quantifies all resource usage and waste generation associated with providing specific goods or services to society. LCIs are increasingly used by industry as well as policy makers to provide a holistic ‘macro’ overview of the environmental profile of a good or service. This information, effectively combined with relevant information obtained from other environmental management tools, is very useful in guiding strategic environmental decision making. LCIs are very data intensive. There is a risk that they imply a level of accuracy that does not exist. This is especially true today, because the availability of accurate LCI data is limited. Also, it is not easy for LCI users, decision-makers and other interested parties to differentiate between ‘good quality’ and ‘poor quality’ LCI data. Several data quality requirements for ‘good’ LCI data can be defined only in relation to the specific study in which they are used. In this paper we show how and why the use of a common LCI database for some of the more commonly used LCI data, together with increased documentation and harmonisation of the data quality features of all LCI data, is key to the further development of LCI as a useful and pragmatic environmental management tool. Initiatives already underway to make this happen are also described.     

Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli

The putative nucleolus in Saccharomyces cerevisiae is visible in electron micrographs as a darkly stained, crescent-shaped structure associated with the nuclear envelope. The haploid yeast genome contains 100 200 tandem copies of a 9.1 kb ribosomal DNA (rDNA) repeat predicted to reside in this structure. We combined in situ hybridization of non-isotopically labeled probes to isolated S. cerevisiae nuclei with immunogold detection to localize rDNA and rRNA precursor sequences in nuclei at the electron microscope (EM) level. Gold particles are restricted to defined regions of nuclei which appear more electron dense than the bulk of the nucleus and which generally exhibit the crescent shape typical of the structure thought to be the nucleolus. In addition, snR17, the yeast homolog of mammalian U3, a nucleolar-restricted small nuclear RNA (snRNA), was localized to the same electron dense region of the nucleus. These data, in conjunction with published immunofluorescent localizations of nucleolarassociated antigens, provide definitive proof that the dense crescent is the nucleolus. Finally, the technique described is applicable to probing nuclear organization in a genetically manipulable system.Abbreviations snRNA small nuclear RNA - AAF N-acetoxy-2-acetyl-aminofluorence by M.L. Pardue     

Analytical tools for characterizing heterogeneity in organelle content

Heterogeneity in the content and function of subcellular organelles on the intercellular and intracellular level plays an important role in determining cell fate. These variations extend to normal-state and disease-state cellular functions and responses to environmental stimuli, such as oxidative stress and therapeutic drugs. Analytical tools to characterize variation in all types of organelles are essential to provide insights that can lead to advances in medicine, such as therapies targeted to specific subcellular regions. In this review, we discuss analytical techniques for interrogating individual intact organelles (e.g. mitochondria and synaptic vesicles) and lysates in a high-throughput manner, including a recently developed nanoscale fluorescence-activated subcellular sorter and techniques based on capillary electrophoresis with laser-induced fluorescence detection. We then highlight the advantages that droplet microfluidics offers for probing subcellular heterogeneity.     

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).