1,721,251 research outputs found
Changes in Chromatin Compaction During the Cell Cycle Revealed by Micrometer-Scale Measurement of Molecular Flow in the Nucleus
Full text linkAbstractWe present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca2+ intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes
Development of an image Mean Square Displacement (iMSD)-based method as a novel approach to study the intracellular trafficking of nanoparticles
Full text linkFluorescence microscopy and spectroscopy techniques are commonly used to investigate complex and interacting biological systems (e.g. proteins and nanoparticles in living cells), since these techniques can explore intracellular dynamics with high time resolution at the nanoscale. Here we extended one of the Image Correlation Spectroscopy (ICS) methods, i.e. the image Mean Square Displacement, in order to study 2-dimensional diffusive and flow motion in confined systems, whose driving speed is uniformly distributed in a variable angular range. Although these conditions are not deeply investigated in the current literature, they can be commonly found in the intracellular trafficking of nanocarriers, which diffuse in the cytoplasm and/or may move along the cytoskeleton in different directions. The proposed approach could reveal the underlying system's symmetry using methods derived from fluorescence correlation concepts and could recover dynamic and geometric features which are commonly done by single particle analyses. Furthermore, it improves the characterization of low-speed flow motions, when compared to SpatioTemporal Image Correlation Spectroscopy (STICS). Although we present a specific example (lipoplexes in living cells), the emphasis is in the discussion of the method, its basic assumptions and its validation on numeric simulations. Statement of Significance Recent advances in nanoparticle-based drug and gene delivery systems have pointed out the interactions at cellular and subcellular levels as key-factors for the efficiency of the adopted biomaterials. Such biochemical and biophysical interactions drive and affect the intracellular dynamics, that is commonly characterized by means of fluorescence microscopy and spectroscopy techniques. Here we present a novel Image Correlation Spectroscopy (ICS) method as a promising tool to capture the intracellular behavior of nanoparticles with high resolution and low background's sensitivity. This study overcomes some of the approximations adopted so far, by decoupling the flow terms of the investigated dynamics and thus recovering ensemble's information from specific single particle behaviors. Finally, relevant implications for nanoparticle-based drug delivery are shown
In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow
Full text linkNo methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here, we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase nucleus of live cells. In the pCF method, we cross-correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of ≈300 ms, suggesting intrinsic localized change in chromatin structure. This is a unique in vivo demonstration of the intricate chromatin network showing channel directed diffusion of an inert molecule with high spatial and temporal resolution.</jats:p
Pair correlation microscopy of intracellular molecular transport
Full text linkPair correlation microscopy is a unique approach to fluorescence correlation spectroscopy that can track the long-range diffusive route of a population of fluorescent molecules in live cells with respect to intracellular architecture. This method is based on the use of a pair correlation function (pCF) that, through spatiotemporal comparison of fluctuations in fluorescence intensity recorded throughout a microscope data acquisition, enables changes in a molecule's arrival time to be spatially mapped and statistically quantified. In this protocol, we present guidelines for the measurement and analysis of line scan pair correlation microscopy data acquired on a confocal laser scanning microscope (CLSM), which will enable users to extract a fluorescent molecule's transport pattern throughout a living cell, and then quantify the molecular accessibility of intracellular barriers encountered or the mode of diffusion governing a molecular trafficking event. Finally, we demonstrate how this protocol can be extended to a two-channel line scan acquisition that, when coupled with a cross pCF calculation, enables a fluorescent molecule's transport pattern to be selectively tracked as a function of complex formation with a spectrally distinct fluorescent ligand. For a skilled user of a CLSM, the line scan data acquisition and analysis described in this protocol will take ~1-2 d, depending on the sample and the number of experiments to be processed
The Impact of Mitotic versus Interphase Chromatin Architecture on the Molecular Flow of EGFP by Pair Correlation Analysis
Full text linkAbstractHere we address the impact nuclear architecture has on molecular flow within the mitotic nucleus of live cells as compared to interphase by the pair correlation function method. The mitotic chromatin is found to allow delayed but continuous molecular flow of EGFP in and out of a high chromatin density region, which, by pair correlation function analysis, is shown as a characteristic arc shape that appears upon entry and exit. This is in contrast to interphase chromatin, which regulates flow between different density chromatin regions by means of a mechanism which turns on and off intermittently, generating discrete bursts of EGFP. We show that the interphase bursts are maintained by metabolic energy, whereas the mitotic mechanism of regulation responsible for the arc is not sensitive to ATP depletion. These two distinct routes of molecular flow were concomitantly measured in the Caenorhabditis elegans germ line, which indicates a conservation of mechanism on a scale more widespread than cell type or organism
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Multiphoton imaging and phasor approach to identify new biomarkers in Huntington Disease
Full text linkNeurodegenerative diseases occur when brain cells (neurons) start to deteriorate. Changes in these cells will lead to dysfunction and eventual cell death. This will lead to mild symptoms like problems with coordination, psychiatric disorders, or memory loss; and as more neurons die the symptoms also progressively worsen. World Health Organization (WHO) indicates up to 1 billion people worldwide are affected by various types of neurodegenerative diseases. In this study, I focused on Huntington disease (HD), a model to study neurodegeneration that is caused by a glitch in a single gene called huntingtin gene (HTT). Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. We all carry HTT; however, the normal length of DNA trinucleotide, CAG, that codes for glutamine are between 10-35. The expanded repeats of above 40 or more will lead to HD. Using advanced functional imaging technique called Two-Photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), and spectral and temporal phasor approach, spectro-temporal phasor map in living mammalian cells and animal tissue was obtained. Using this sophisticated imaging technique, I have developed new methods and identified novel biomarkers that can help detect Huntington disease early on. This can also help for evaluating the efficacy of treatment. The novel method established in this work is noninvasive and can be performed at the single cell level. Phasor transformation used here simplifies the FLIM and spectral measurements by providing a graphical global view of the process at each pixel and avoids some of the complexity of the multi-exponential analysis. In this way, using a fit free approach that can be applied to both time and frequency domain measurements, Fluorescence Lifetime and spectral emission can be analyzed. It is hoped that this work shed a light on understanding the mechanism of Huntington disease and for new drug discovery and early diagnosis of the disease. The approach introduced in this work can also be applied as a method for understating similar neurodegenerative diseases. This work is supported in part by NIH grant P41 GM103540, NSF BEST IGERT and UC PDY grant
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Amyloid-β(1-42) Peptide Induces Neutral Lipids Accumulation in Hippocampal Neurons in Normal and Alzheimer’s Disease Aging Models, Lipid Environment Remodeling Revealed by Hyperspectral Imaging and the Phasor Approach
Full text linkAmyloid-β (Aβ), particularly the Aβ(1-42) variant, plays dual roles in neuronal physiology and pathology. While essential for synaptic function at physiological levels, its accumulation induces oxidative stress, membrane disruption, and lipid peroxidation, contributing to Alzheimer’s disease (AD). Aβ interacts with lipid rafts, disturbing membrane composition and promoting further Aβ production. Lipid droplets (LDs), key in lipid storage, lipid transport and stress response, are altered by Aβ, showing impaired mobilization and increased abundance. This study uses nile red solvatochromic properties and hyperspectral imaging to analyze the lipid polarity of hippocampal neurons and LDs dynamics in neurons exposed to exogenous-labeled Aβ(1-42) (exAβ), revealing age- and genotype-dependent lipid remodeling in normal- and AD-aging.We employed hyperspectral confocal imaging and advanced image analysis to map lipid organization in primary hippocampal neurons from non-transgenic (NTg) and triple-transgenic (3xTg) AD mice in response to exAβ-mediated stress, using nile red, a lipophilic and environment sensitive fluorescent dye. The spectral phasor transformation converted each pixel’s emission spectrum into G and S coordinates, enabling unmixing of fluorescently tagged exAβ, and Nile Red’s neutral and polar lipid signals without fitting models. Custom segmentation generated binary masks for whole-cell, plasma membrane, cytosol, lipid droplets and vesicular structures containing exAβ (Ves-exAβ+), by thresholding channel projections and applying morphological operations. For spatial analysis, Phasor analysis of Local Image Correlation Spectroscopy (PLICS) computed local autocorrelation functions over m×m subimages, then applied Fourier phasor transforms to extract real size and clustering metrics of LDs and Ves-exAβ+. This combined pipeline quantified compartment-specific lipid polarity indices, droplet number, size distributions, and spatial relationships, revealing age and genotype-dependent lipid remodeling under exogenous Aβ(1-42) exposure.Hyperspectral confocal imaging with three-component spectral phasor analysis and PLICS revealed that both, genotype and exogenous Aβ(1-42), independently remodel lipid organization in hippocampal neurons. Our results link higher exAβ accumulation to lower lipid polarity, genotype-driven polarity differences and age-dependent increases in lipid droplet (LD) and Ves-exAβ+ counts. In whole-cell, cytosol, and membrane compartments, NTg neurons show steadily rising lipid polarity profiles from young to middle age, whereas 3xTgAD cells peak at middle age then decline, with exAβ accelerating polarity loss in both. LD analysis demonstrated that NTg LD counts rise then fall with age but are amplified by exAβ, with small LDs shrinking from ~81% to ~29% and large LDs enlarging to ~71%, while 3xTg-AD neurons exhibit early LD enlargement and altered clustering under exAβ stress. Finally, Ves-exAβ+ structures increase with age—more abruptly in 3xTgAD—cluster more tightly or fuse in old cells, and shift toward larger Aβ-rich vesicles, whose lipid polarity varies by size and genotype, highlighting complex, age and genotype-dependent Aβ handling.Together, these results demonstrate that exogenous Aβ(1-42) and AD genotype independently drive compartment-specific lipid remodeling, they jointly accelerate polarity loss in the membrane, cytosol, and LD pools, amplifying LD number, size, and clustering, and promoting the formation of larger, more hydrophobic Aβ-positive vesicles, highlighting dynamic lipid–Aβ codependency in aging neurons
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Fluorescence Lifetime Imaging Microscopy and LAURDAN Spectral Imaging for Dynamically Investigating Osteoclast Differentiation
Full text linkOsteoclasts, the multinucleated bone-resorbing cells, are involved in the destructive breakdown of bones in many diseases such as osteoporosis and rheumatoid arthritis. Designing an efficient and specific therapeutic strategy to these diseases would depend on understanding osteoclasts differentiation. Although gene expression quantification and biochemical techniques have been used extensively to study osteoclast differentiation, they lack the capability to dynamically examine live osteoclasts at the single-cell level. In this thesis, we explored the practicality of the two minimally invasive microscopy techniques, NAD(P)H Fluorescence Lifetime Imaging and LAURDAN spectral imaging, in observing cellular metabolic profiles and membrane dynamics respectively during osteoclast differentiation. In addition to establishing the practicality of these two imaging platforms, our report offered a deeper understanding regarding the roles of metabolism and membrane dynamics in osteoclasts differentiation and pathogenesis of osteoclasts-associated diseases
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Assessment of Embryo Health and Circulating Tumor Cell Metabolism Using the Phasor-FLIM Approach
Full text linkCellular functional and structural changes associated with metabolism are essential for understanding healthy tissue development and the progression of numerous diseases. Quantitatively monitoring of metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of the diseases. Unfortunately, established methods for this purpose are either destructive or require the use of exogenous agents. Recent work has highlighted the potential of endogenous two-photon excited fluorescence as a method to monitor subtle metabolic changes. In this thesis, we apply two-photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic imaging in pre-implantation embryos and other biological samples.We exploited the intrinsically fluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. Using phasor-FLIM, we identified unique metabolic states that distinguish embryonic stem cells from differentiating progeny. We also apply the phasor-FLIM and hyperspectral microscopy to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological and non-invasive caliber for embryo quality. We identify the unique spectroscopic trajectories at different stages of mouse pre-implantation development which can be further used to distinguish pre-implantation embryo quality using an artificial intelligence algorithm at the early compaction stage with 86% accuracy. Furthermore, we showed the heterogeneity and changes in the normal pre-implantation embryos and aneuploidy embryos treated with the spindle assembly checkpoint inhibitor during embryo division can be rapidly distinguished at blastocyst stage via spectra phasor. Finally, we designed rapid and label-free single leukemia cell identification platform that combines high-throughput size-based separation of hemocytes, and leukemia cell identification through phasor approach and phasor-FLIM to quantify changes between free/bound NADH as an indirect measurement of metabolic alteration in living cells.These examples illustrate the potential of fluorescence lifetime imaging microscopy for unveiling complex physiological processes. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine
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Analyses of the Cellular Signaling Responses to DNA Damage and DNA Repair Factor Recruitment Using Fluorescence Lifetimes and Fluctuations
Full text linkGenome integrity is continually challenged by threats including DNA replication errors, toxic metabolic byproducts, and exposure to exogenous genotoxins. Responding to and repairing damaged DNA requires coordinating a number of critical cellular events including activating DNA repair, facilitating chromatin rearrangement, and delaying cell cycle progression. Although the factors important for DNA repair have been identified, how these activities are coordinated in the nucleus and the long-term cellular-wide consequences of DNA repair are not well characterized. Nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in both cellular metabolism and DNA repair. Thus, analyses of NAD species could provide new insight into DNA damage signaling as well as damage-induced metabolic responses to various types of DNA lesions. Here, we use the phasor approach to fluorescence lifetime imaging microscopy in combination with genetically encoded fluorescent biosensors to measure metabolic changes in single cells with high spatiotemporal resolution. We found that the absence of the reversionless 3-like translesional synthesis DNA repair protein promotes p53-mediated upregulation of oxidative phosphorylation (oxphos) in cisplatin-treated H1299 lung carcinoma cells and increases cell sensitivity to this chemotherapeutic treatment. Furthermore, it has been well-documented that depletion of NAD+ leads to an overall metabolic collapse, but it is not clear whether or not regulating metabolism can overcome cell death pathways as a survival mechanism. We observe a PARP-dependent decrease in NAD species and an increased metabolic reliance on oxphos. In all, our analyses revealed a previously unrecognized long-term effect of DNA repair signaling on energy metabolism in DNA damaged cells. Although the patterns of DNA repair protein redistribution following DNA damage have been systematically documented, understanding the complex response to damage requires the characterization of the molecular dynamics of these proteins with high spatiotemporal resolution. By using spatial pair cross-correlation function analysis in two-dimensions, we were able to visualize the barriers to molecular motion of DNA repair proteins in response to laser microirradiation-induced DNA damage.In summary, my project utilized cutting-edge fluorescence dynamics techniques to reveal a connection between the DNA damage response and cellular metabolism and to develop a new method to characterize molecular diffusion in response to DNA damage
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