Non‐linear microscopy is a powerful imaging tool to examine structural properties and subcellular... more Non‐linear microscopy is a powerful imaging tool to examine structural properties and subcellular processes of various biological samples. The competence of Third Harmonic Generation (THG) includes the label free imaging with diffraction‐limited resolution and three‐ dimensional visualization with negligible phototoxicity effects. In this study, THG records and quantifies the lipid content of Drosophila haemocytes, upon encountering normal or tumorigenic neural cells, in correlation with their shape or their state. We show that the lipid accumulations of adult haemocytes are similar before and after encountering normal cells. In contrast, adult haemocytes prior to their interaction with cancer cells have a low lipid index, which increases while they are actively engaged in phagocytosis only to decrease again when haemocytes become exhausted. This dynamic change in the lipid accrual of haemocytes upon encountering tumour cells could potentially be a useful tool to assess the phagocyt...
Aging is the major risk factor for several life‐threatening pathologies and impairs the function ... more Aging is the major risk factor for several life‐threatening pathologies and impairs the function of multiple cellular compartments and organelles. Age‐dependent deterioration of nuclear morphology is a common feature in evolutionarily divergent organisms. Lipid droplets have been shown to localize in most nuclear compartments, where they impinge on genome architecture and integrity. However, the significance of progressive nuclear lipid accumulation and its impact on organismal homeostasis remain obscure. Here, we implement non‐linear imaging modalities to monitor and quantify age‐dependent nuclear lipid deposition in Caenorhabditis elegans. We find that lipid droplets increasingly accumulate in the nuclear envelope, during aging. Longevity‐promoting interventions, such as low insulin signaling and caloric restriction, abolish the rate of nuclear lipid accrual and decrease the size of lipid droplets. Suppression of lipotoxic lipid accumulation in hypodermal and intestinal nuclei is ...
The cell membrane is a highly complex structure consisting of a large diversity of phospholipids ... more The cell membrane is a highly complex structure consisting of a large diversity of phospholipids and macromolecules. There exist a variety of diseases that compromise the integrity of this key component of the cell. This thesis considers the investigation of interactions between β-amyloid peptide (Aβ42) and lipid bilayers. To facilitate understanding of this complex system, it is advantageous to employ a model sample; supported lipid bilayers (SLB) and giant multilamellar vesicles (MLVs) are used as proxy cell membranes. These nanostructures are widely used as models of cellular membranes in many areas of scientific research. Phospholipid molecules self-organise into bilayer structures containing phase-separated microdomains, which are believed to be important in many biological processes. This study aims to develop model systems and experimental tools to explore hypothetical mechanisms through which the β-amyloid interacts with the lipid membranes. A lack of mechanistic understandi...
The in-vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging v... more The in-vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging via non-invasive label free imaging techniques is an important issue with high biological significance. In the present study, polarization-dependent second-harmonic generation (PSHG) was used to evaluate structural alterations in the striated muscles during Caenorhabditis elegans lifespan. Young and old wild type animals were irradiated. The obtained results showed that it was not feasible to detect differences in the structure of myosin that could be correlated with the aging of the worms, via the implementation of the classical, widely used, cylindrical symmetry model and the calculation of the SHG anisotropy values. A trigonal symmetry model improved the extracted results, however the best outcome originated from the application of a general model. Myosin structural modifications were monitored via the estimation of the difference in spectral phases derived from Discrete Fourier Transform analysis. Age classification of the nematodes was achieved by employing both models, proving the usefulness of the usage of PSHG microscopy as a potential diagnostic tool for the investigation of muscle diseases. This article is protected by copyright. All rights reserved.
A range of sophisticated imaging techniques have been developed in recent years that can reveal t... more A range of sophisticated imaging techniques have been developed in recent years that can reveal the surface structure of cultural heritage objects with varying precision. In combination with various spectroscopic methods, they allow the study of the chemical composition of the object; thus, conclusions can be drawn about the origin of the object or its initial components, method, or time of creation, authenticity, mechanisms of degradation, and ways of further conservation. At present, different techniques can be applied to a wide range of cultural heritage objects, such as varnishes, paintings, archaeological objects, binding media, paper-based documents, parchments, marbles, frescoes, as well as various objects made of leather, fabric, stone, ceramics and glass, wood, or metal. One of the main needs in the study of cultural heritage (CH) is the transportability/portability of the research equipment, since many pieces under investigation cannot be moved to the laboratory, either be...
The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a... more The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a complex of pathological conditions including type-2 diabetes, hypertension, and cardiovascular diseases, is an important issue with high biological significance and requires accurate methods capable of monitoring lipid storage distribution and dynamics in vivo. In this study, the nonlinear phenomena of second and third harmonic generation (SHG, THG) have been employed simultaneously as label-free, nondestructive diagnostic techniques, for the monitoring and the complementary three-dimensional (3D) imaging and analysis of the muscular areas and the lipid content localization. THG microscopy was used as a quantitative tool in order to record the accumulation of lipids in nonadipose tissues in the pharyngeal muscles of 18 Caenorhabditis elegans (C. elegans) specimens, while the SHG imaging provided the detailed anatomical information about the structure of the muscles. The ectopic accumula...
Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat depo... more Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat deposition in nonadipose tissues. Ectopic fat contributes to lipotoxicity and has been implicated in the development of a metabolic syndrome that increases risk of age-associated diseases. However, the molecular mechanisms coupling ectopic fat accumulation with aging remain obscure. Here, we use nonlinear imaging modalities to visualize and quantify age-dependent ectopic lipid accumulation in Caenorhabditis elegans We find that aging is accompanied by pronounced deposition of lipids in nonadipose tissues, including the nervous system. Importantly, interventions that promote longevity such as low insulin signaling, germ-line loss, and dietary restriction, which effectively delay aging in evolutionary divergent organisms, diminish the rate of ectopic fat accumulation and the size of lipid droplets. Suppression of lipotoxic accumulation of fat in heterologous tissues is dependent on helix-loop-...
Advanced Microscopy Techniques IV; and Neurophotonics II, 2015
The non-linear techniques Second and Third Harmonic Generation (SHG, THG) have been employed simu... more The non-linear techniques Second and Third Harmonic Generation (SHG, THG) have been employed simultaneously to record three dimensional (3D) imaging and localize the lipid content of the muscular areas (ectopic fat) of Caenorhabditis elegans (C. elegans). Simultaneously, Two-Photon Fluorescence (TPEF) was used initially to localize the stained lipids with Nile Red, but also to confirm the THG potential to image lipids successfully. In addition, GFP labelling of the somatic muscles, proves the initial suggestion of the existence of ectopic fat on the muscles and provides complementary information to the SHG imaging of the pharynx. The ectopic fat may be related to a complex of pathological conditions including type-2 diabetes, hypertension and cardiovascular diseases. The elucidation of the molecular path leading to the development of metabolic syndrome is a vital issue with high biological significance and necessitates accurate methods competent of monitoring lipid storage distribution and dynamics in vivo. THG microscopy was employed as a quantitative tool to monitor the lipid accumulation in non-adipose tissues in the pharyngeal muscles of 12 unstained specimens while the SHG imaging revealed the anatomical structure of the muscles. The ectopic fat accumulation on the pharyngeal muscles increases in wild type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of the ectopic fat accumulation with the aging. Our results can provide new evidence relating the deposition of ectopic fat with aging, but also validate SHG and THG microscopy modalities as new, non-invasive tools capable of localizing and quantifying selectively lipid accumulation and distribution.
ABSTRACT We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging o... more ABSTRACT We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging on which we combine Coherent Anti-Stokes Raman Scattering (CARS) with Two Photon Excitation Fluorescence (TPEF) using a Nd: YVO4 pump laser. We acquired 2PEF, CARS, and phase contrast images of Multilamellar Vesicles (MLVs) and Giant Unilamellar Vesicles (GUVs), as well as Raman spectra of the constituent lipids. A wide range of peptides are harmful to cells by altering the structure of the biological membranes. This effect depends on the composition of the membrane and the chemical structure of the peptide. The peptide we studied is the beta amyloid Aβ which is a major component of the amyloid plaques deposited on neuronal membranes of Alzheimer's disease (AD) patients. AD is neurodegenerative disorder in which the hallmark symptoms include cognitive decline and dementia[2] and is characterized by the formation of extracellular amyloid fibrils on the neuronal membranes of the brain. Many questions still remain unanswered concerning the destabilization of cellular ionic homeostasis due to pores formed during the interactions of lipid membranes with peptides. In this project, biomimics of cell membranes are used. The structures that best mimic the plasma membranes are MLVs or GUVs. These vesicles are formed using the gentle hydration technique[3] or the electroformation technique[4] respectively and are composed of phospholipids such as DOPC, DPPC, D62PPC and their binary mixtures. The MLVs and GUVs imaging by CARS and TPEF microscopy not only permits the direct imaging of the leakage phenomenon caused by the toxic peptide (Aβ) on the lipid bilayer, but also records simultaneously the lateral structure of the bilayer and peptide distribution in the plane across the membrane.
Non‐linear microscopy is a powerful imaging tool to examine structural properties and subcellular... more Non‐linear microscopy is a powerful imaging tool to examine structural properties and subcellular processes of various biological samples. The competence of Third Harmonic Generation (THG) includes the label free imaging with diffraction‐limited resolution and three‐ dimensional visualization with negligible phototoxicity effects. In this study, THG records and quantifies the lipid content of Drosophila haemocytes, upon encountering normal or tumorigenic neural cells, in correlation with their shape or their state. We show that the lipid accumulations of adult haemocytes are similar before and after encountering normal cells. In contrast, adult haemocytes prior to their interaction with cancer cells have a low lipid index, which increases while they are actively engaged in phagocytosis only to decrease again when haemocytes become exhausted. This dynamic change in the lipid accrual of haemocytes upon encountering tumour cells could potentially be a useful tool to assess the phagocyt...
Aging is the major risk factor for several life‐threatening pathologies and impairs the function ... more Aging is the major risk factor for several life‐threatening pathologies and impairs the function of multiple cellular compartments and organelles. Age‐dependent deterioration of nuclear morphology is a common feature in evolutionarily divergent organisms. Lipid droplets have been shown to localize in most nuclear compartments, where they impinge on genome architecture and integrity. However, the significance of progressive nuclear lipid accumulation and its impact on organismal homeostasis remain obscure. Here, we implement non‐linear imaging modalities to monitor and quantify age‐dependent nuclear lipid deposition in Caenorhabditis elegans. We find that lipid droplets increasingly accumulate in the nuclear envelope, during aging. Longevity‐promoting interventions, such as low insulin signaling and caloric restriction, abolish the rate of nuclear lipid accrual and decrease the size of lipid droplets. Suppression of lipotoxic lipid accumulation in hypodermal and intestinal nuclei is ...
The cell membrane is a highly complex structure consisting of a large diversity of phospholipids ... more The cell membrane is a highly complex structure consisting of a large diversity of phospholipids and macromolecules. There exist a variety of diseases that compromise the integrity of this key component of the cell. This thesis considers the investigation of interactions between β-amyloid peptide (Aβ42) and lipid bilayers. To facilitate understanding of this complex system, it is advantageous to employ a model sample; supported lipid bilayers (SLB) and giant multilamellar vesicles (MLVs) are used as proxy cell membranes. These nanostructures are widely used as models of cellular membranes in many areas of scientific research. Phospholipid molecules self-organise into bilayer structures containing phase-separated microdomains, which are believed to be important in many biological processes. This study aims to develop model systems and experimental tools to explore hypothetical mechanisms through which the β-amyloid interacts with the lipid membranes. A lack of mechanistic understandi...
The in-vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging v... more The in-vivo elucidation of the molecular mechanisms underlying muscles dysfunction due to aging via non-invasive label free imaging techniques is an important issue with high biological significance. In the present study, polarization-dependent second-harmonic generation (PSHG) was used to evaluate structural alterations in the striated muscles during Caenorhabditis elegans lifespan. Young and old wild type animals were irradiated. The obtained results showed that it was not feasible to detect differences in the structure of myosin that could be correlated with the aging of the worms, via the implementation of the classical, widely used, cylindrical symmetry model and the calculation of the SHG anisotropy values. A trigonal symmetry model improved the extracted results, however the best outcome originated from the application of a general model. Myosin structural modifications were monitored via the estimation of the difference in spectral phases derived from Discrete Fourier Transform analysis. Age classification of the nematodes was achieved by employing both models, proving the usefulness of the usage of PSHG microscopy as a potential diagnostic tool for the investigation of muscle diseases. This article is protected by copyright. All rights reserved.
A range of sophisticated imaging techniques have been developed in recent years that can reveal t... more A range of sophisticated imaging techniques have been developed in recent years that can reveal the surface structure of cultural heritage objects with varying precision. In combination with various spectroscopic methods, they allow the study of the chemical composition of the object; thus, conclusions can be drawn about the origin of the object or its initial components, method, or time of creation, authenticity, mechanisms of degradation, and ways of further conservation. At present, different techniques can be applied to a wide range of cultural heritage objects, such as varnishes, paintings, archaeological objects, binding media, paper-based documents, parchments, marbles, frescoes, as well as various objects made of leather, fabric, stone, ceramics and glass, wood, or metal. One of the main needs in the study of cultural heritage (CH) is the transportability/portability of the research equipment, since many pieces under investigation cannot be moved to the laboratory, either be...
The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a... more The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a complex of pathological conditions including type-2 diabetes, hypertension, and cardiovascular diseases, is an important issue with high biological significance and requires accurate methods capable of monitoring lipid storage distribution and dynamics in vivo. In this study, the nonlinear phenomena of second and third harmonic generation (SHG, THG) have been employed simultaneously as label-free, nondestructive diagnostic techniques, for the monitoring and the complementary three-dimensional (3D) imaging and analysis of the muscular areas and the lipid content localization. THG microscopy was used as a quantitative tool in order to record the accumulation of lipids in nonadipose tissues in the pharyngeal muscles of 18 Caenorhabditis elegans (C. elegans) specimens, while the SHG imaging provided the detailed anatomical information about the structure of the muscles. The ectopic accumula...
Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat depo... more Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat deposition in nonadipose tissues. Ectopic fat contributes to lipotoxicity and has been implicated in the development of a metabolic syndrome that increases risk of age-associated diseases. However, the molecular mechanisms coupling ectopic fat accumulation with aging remain obscure. Here, we use nonlinear imaging modalities to visualize and quantify age-dependent ectopic lipid accumulation in Caenorhabditis elegans We find that aging is accompanied by pronounced deposition of lipids in nonadipose tissues, including the nervous system. Importantly, interventions that promote longevity such as low insulin signaling, germ-line loss, and dietary restriction, which effectively delay aging in evolutionary divergent organisms, diminish the rate of ectopic fat accumulation and the size of lipid droplets. Suppression of lipotoxic accumulation of fat in heterologous tissues is dependent on helix-loop-...
Advanced Microscopy Techniques IV; and Neurophotonics II, 2015
The non-linear techniques Second and Third Harmonic Generation (SHG, THG) have been employed simu... more The non-linear techniques Second and Third Harmonic Generation (SHG, THG) have been employed simultaneously to record three dimensional (3D) imaging and localize the lipid content of the muscular areas (ectopic fat) of Caenorhabditis elegans (C. elegans). Simultaneously, Two-Photon Fluorescence (TPEF) was used initially to localize the stained lipids with Nile Red, but also to confirm the THG potential to image lipids successfully. In addition, GFP labelling of the somatic muscles, proves the initial suggestion of the existence of ectopic fat on the muscles and provides complementary information to the SHG imaging of the pharynx. The ectopic fat may be related to a complex of pathological conditions including type-2 diabetes, hypertension and cardiovascular diseases. The elucidation of the molecular path leading to the development of metabolic syndrome is a vital issue with high biological significance and necessitates accurate methods competent of monitoring lipid storage distribution and dynamics in vivo. THG microscopy was employed as a quantitative tool to monitor the lipid accumulation in non-adipose tissues in the pharyngeal muscles of 12 unstained specimens while the SHG imaging revealed the anatomical structure of the muscles. The ectopic fat accumulation on the pharyngeal muscles increases in wild type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of the ectopic fat accumulation with the aging. Our results can provide new evidence relating the deposition of ectopic fat with aging, but also validate SHG and THG microscopy modalities as new, non-invasive tools capable of localizing and quantifying selectively lipid accumulation and distribution.
ABSTRACT We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging o... more ABSTRACT We have used a versatile and powerful microscope[1] for multi-modal biomedical imaging on which we combine Coherent Anti-Stokes Raman Scattering (CARS) with Two Photon Excitation Fluorescence (TPEF) using a Nd: YVO4 pump laser. We acquired 2PEF, CARS, and phase contrast images of Multilamellar Vesicles (MLVs) and Giant Unilamellar Vesicles (GUVs), as well as Raman spectra of the constituent lipids. A wide range of peptides are harmful to cells by altering the structure of the biological membranes. This effect depends on the composition of the membrane and the chemical structure of the peptide. The peptide we studied is the beta amyloid Aβ which is a major component of the amyloid plaques deposited on neuronal membranes of Alzheimer's disease (AD) patients. AD is neurodegenerative disorder in which the hallmark symptoms include cognitive decline and dementia[2] and is characterized by the formation of extracellular amyloid fibrils on the neuronal membranes of the brain. Many questions still remain unanswered concerning the destabilization of cellular ionic homeostasis due to pores formed during the interactions of lipid membranes with peptides. In this project, biomimics of cell membranes are used. The structures that best mimic the plasma membranes are MLVs or GUVs. These vesicles are formed using the gentle hydration technique[3] or the electroformation technique[4] respectively and are composed of phospholipids such as DOPC, DPPC, D62PPC and their binary mixtures. The MLVs and GUVs imaging by CARS and TPEF microscopy not only permits the direct imaging of the leakage phenomenon caused by the toxic peptide (Aβ) on the lipid bilayer, but also records simultaneously the lateral structure of the bilayer and peptide distribution in the plane across the membrane.
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record three dimensional (3D) imaging and localize the lipid content of the muscular areas (ectopic fat) of Caenorhabditis
elegans (C. elegans). Simultaneously, Two-Photon Fluorescence (TPEF) was used initially to localize the stained lipids
with Nile Red, but also to confirm the THG potential to image lipids successfully. In addition, GFP labelling of the somatic
muscles, proves the initial suggestion of the existence of ectopic fat on the muscles and provides complementary
information to the SHG imaging of the pharynx. The ectopic fat may be related to a complex of pathological conditions
including type-2 diabetes, hypertension and cardiovascular diseases. The elucidation of the molecular path leading to the
development of metabolic syndrome is a vital issue with high biological significance and necessitates accurate methods
competent of monitoring lipid storage distribution and dynamics in vivo. THG microscopy was employed as a quantitative
tool to monitor the lipid accumulation in non-adipose tissues in the pharyngeal muscles of 12 unstained specimens while
the SHG imaging revealed the anatomical structure of the muscles. The ectopic fat accumulation on the pharyngeal muscles
increases in wild type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of the ectopic fat
accumulation with the aging. Our results can provide new evidence relating the deposition of ectopic fat with aging, but
also validate SHG and THG microscopy modalities as new, non-invasive tools capable of localizing and quantifying
selectively lipid accumulation and distribution.
record three dimensional (3D) imaging and localize the lipid content of the muscular areas (ectopic fat) of Caenorhabditis
elegans (C. elegans). Simultaneously, Two-Photon Fluorescence (TPEF) was used initially to localize the stained lipids
with Nile Red, but also to confirm the THG potential to image lipids successfully. In addition, GFP labelling of the somatic
muscles, proves the initial suggestion of the existence of ectopic fat on the muscles and provides complementary
information to the SHG imaging of the pharynx. The ectopic fat may be related to a complex of pathological conditions
including type-2 diabetes, hypertension and cardiovascular diseases. The elucidation of the molecular path leading to the
development of metabolic syndrome is a vital issue with high biological significance and necessitates accurate methods
competent of monitoring lipid storage distribution and dynamics in vivo. THG microscopy was employed as a quantitative
tool to monitor the lipid accumulation in non-adipose tissues in the pharyngeal muscles of 12 unstained specimens while
the SHG imaging revealed the anatomical structure of the muscles. The ectopic fat accumulation on the pharyngeal muscles
increases in wild type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of the ectopic fat
accumulation with the aging. Our results can provide new evidence relating the deposition of ectopic fat with aging, but
also validate SHG and THG microscopy modalities as new, non-invasive tools capable of localizing and quantifying
selectively lipid accumulation and distribution.