Filippidis George

• Foundation for Research and Technology Hellas

Second‐harmonic generation (SHG) microscopy provides a high‐resolution label‐free approach for noninvasively detecting collagen organization and its pathological alterations. Up to date, several imaging analysis algorithms for extracting collagen morphological features from SHG images—such as fiber size and length, order and anisotropy—have been de...

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, TH...

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...

The availability of non-invasive technologies, which can be used separately or in combination for obtaining chemical composition data and structural information of Cultural Heritage (CH) materials, is of prime importance for improving the understanding the environmental or ageing impact on monuments and artefacts and defining optimal strategies for...

Whispering gallery mode (WGM) resonances in radially, thermally poled glass hetero-fibers, are investigated for the first time. Upon radially oriented thermal poling, both TE and TM polarized responses undergoing a spectral “cleaning” process, namely higher-order radial modes are being suppressed, or even fully annihilated, compared to the spectral...

Polarization-dependent second harmonic generation (PSHG) microscopy is used as an innovative, high-resolution, non-destructive, and label-free diagnostic imaging tool to elucidate biological issues with high significance. In the present study, information on the structure and directionality of collagen fibers in equine pericardium tissue was collec...

The development of label-free non-invasive techniques to be used as diagnostic tools in cancer research is of great importance for improving the quality of life for millions of patients. Previous studies have demonstrated that Third Harmonic Generation (THG) imaging could differentiate malignant from benign unlabeled human breast biopsies and disti...

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 dur...

Three-dimensional control in the tuning of the linear and nonlinear properties of optical materials can catalyse the design of new photonic devices. Glasses being the backbone materials for drawing optical fibres, are inherently centrosymmetric, so their nonlinear dielectric susceptibility χ(2) is zero; thermal poling has been used for breaking the...

Non‐linear optical imaging techniques have been widely used to reveal biological structures for accurate diagnosis at the cellular as well as the tissue level. In the present study, polarization‐dependent second‐harmonic generation (PSHG) was used to determine collagen orientation in breast cancer biopsy tissues (grades 0, I, II and III). The obtai...

The ability to distinguish and grade malignant cells during surgical procedures in a fast, non-invasive and staining-free manner is of high importance in tumor management. To this extend, Third Harmonic Generation (THG), Second Harmonic Generation (SHG) and Fourier-Transform Infrared (FTIR) spectroscopy were applied to discriminate malignant from h...

A hybrid, microstructured optical fiber (MOF) comprised of a silica glass, endlessly single mode optical fiber infiltrated with 2-methyl, 4-nitroaniline (MNA) is subjected to electric field poling for enhancing non-linear wavelength conversion properties. Different poling conditions (temperature, and electric field) are applied, resulting in signif...

The non-invasive depth-resolved imaging of pictorial layers in paintings by means of linear optical techniques represents a challenge in the field of Cultural Heritage (CH). The presence of opaque and/or highly-scattering materials may obstruct the penetration of the radiation probe, thus impeding the visualization of the stratigraphy of paintings....

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...

Paintings are particularly sensitive and fragile, mainly due to their complexity in materials and structures and their susceptibility to environmental conditions. In this context, the nature and properties of laser light allow for accurate and sensitive measurements, as well as for selective and controlled interventions, by employing unique diagnos...

We demonstrate the effective combination of multiphoton and photoacoustic (PA) imaging for the high-resolution stratigraphic analysis of multilayered art objects with emphasis on paintings. A novel convolution-based algorithm is additionally applied for the precise discrimination of nonlinear signals, providing valuable information in regard to the...

In this paper, the authors present an overview of the implementation of non-linear imaging contrast microscopy techniques as novel, powerful diagnostic tools for cultural heritage (CH) studies. These high-resolution, non-destructive modalities are employed for the in-depth compositional identification and morphological characterisation of protectiv...

The ability to monitor the activation state of T‐cells during immunotherapy is of great importance. Although specific activation markers do exist, their abundance and complicated regulation cannot definitely define the activation state of the cells. Previous studies have shown that Third Harmonic Generation (THG) imaging could distinguish between a...

Implantation failure is a major problem in human assisted reproduction, which persists regardless the optimization of endometrial receptivity and selection of genetically and morphologically healthy embryos. Since embryo-endometrium interaction depends on cell junctional, cell adhesion and cell-substratum adhesion molecules, the present study inqui...

Non-linear optical imaging techniques have been used to greatly enhance our understanding of issues with high biological significance and promise a strong impact on early and accurate detection of various diseases. In our current work, we employ Third Harmonic Generation (THG) and Second Harmonic Generation (SHG) imaging modalities for diagnosis of...

The nonlinear optical microscopy (NLM) modalities of Multi-Photon Excited Fluorescence (MPEF) and Third Harmonic Generation (THG) have been combined in this work to characterize as a function of depth with...

This paper examines for the first time the potential complementary imaging capabilities of Optical coherence tomography (OCT) and non-linear microscopy (NLM) for multi-modal 3D examination of paintings following the successful application of OCT to the in situ, non-invasive examination of varnish and paint stratigraphy of historic paintings and the...

Third Harmonic Generation (THG) imaging was applied to mouse resting and activated T-cells. Quantification of THG signal, which corresponded to lipid droplets, could distinguish activated Tcells, allowing follow-up of immune response development.

Age-dependent collapse of lipid homeostasis results in spillover of lipids and excessive fat deposition in non-adipose 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...

Nonlinear optical imaging techniques are at the forefront of biomedical research over the last years. The ability of label free imaging, diffraction limited resolution, high penetration depth, intrinsic three dimensional (3D) sectioning and low phototoxicity make this technology a perfect diagnostic tool in many biomedical applications. This review...

Third Harmonic Generation (THG) microscopy as a non‐invasive, label free imaging methodology, allows linkage of lipid profiles with various breast cancer cells. The collected THG signal arise mostly from the lipid droplets and the membrane lipid bilayer. Quantification of THG signal can accurately distinguish HER2‐positive cells. Further analysis u...

In this work, we report that polarization second harmonic generation (PSHG) microscopy, commonly used in biomedical imaging, can quantitatively discriminate naturally aged from fresh starch-based glues used for conservation or restoration of paintings, works of art on paper, and books. Several samples of fresh and aged (7 years) flour and starch pa...

L-carnitine (L-Cn) despite the beneficial role as energy generating substance delivering long-chain fatty acids to the β-oxidation pathway in mitochondria has been accused to cause an endometriosis-like state to BALB/c mice manifested by increased inflammatory cytokines in serum and peritoneal fluid, accumulation of immune cells in the peritoneal c...

Nonlinear optical imaging techniques have created new opportunities of research in the biomedical field. Specifically, Third Harmonic Generation (THG) seems to be a suitable noninvasive imaging tool for the delineation and quantification of biological structures at the microscopic level. The aim of this study was to extract information as to the ac...

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 lipi...

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...

Nonlinear optical imaging techniques have created new opportunities of research in the biomedical field. Specifically, Third Harmonic Generation (THG) seems to be a suitable noninvasive imaging tool for the delineation and quantification of biological structures at the microscopic level. The aim of this study was to extract information as to the ac...

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 lipi...

The present work investigates the applicability of nonlinear imaging microscopy for the precise assessment of degradation of the outer protective layers of painted artworks as a function of depth due to aging. Two fresh and artificially aged triterpenoid varnishes, dammar and mastic, were tested. Nonlinear imaging techniques have been employed as a...

Two types of highly fluorescent carbon dots (C-dots) were prepared by a single-step procedure based on microwave heating citric acid and 6-aminocaproic acid or citric acid and urea in an aqueous solution. The small size of the isolated carbon dots along with their strong absorption in the UV and their excitation wavelength-dependent fluorescence re...

While the role of L-carnitine in lipid metabolism is well established, it has also been recognized to affect the female reproductive system. Daily administration of 2.5 mg of L-carnitine to young female mice for 7 days increases the percentage of immune cells in the peritoneal exudates and uterus and the levels of IFN-γ, TNF-α, IL-2, 4, 6, VEGF, GM...

Here, we present a review of the implementa-tion of nonlinear imaging microscopy techniques such as second and third harmonic generation (SHG-THG) and multi-photon excitation fluorescence (MPEF), as high-res-olution, non-invasive diagnostic tools for cultural heritage studies. Specifically, the above nonlinear modalities are employed for the precis...

Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in viv...

Insight into the processes taking place upon ultraviolet (UV) laser ablation of polymers is of high importance. In this study, a new algorithm for the precise analysis of third harmonic generation (THG) images from irradiated polymers has been developed. In particular, high accuracy qualitative and quantitative morphological information concerning...

In this work, nonlinear laser scanning microscopy was employed for the characterization and three-dimensional (3D) imaging of microlenses and microlens arrays. Third-harmonic generation and two-photon excitation fluorescence (TPEF) signals were recorded and the obtained data were further processed in order to generate 3D reconstructions of the exam...

In this study, we present results of the implementation of multi photon excitation fluorescence (MPEF) imaging measurements to silver-based artifacts for the identification of the corrosion layers (generally composed by chlorargyrite, AgCl). We employ as an excitation source a compact femtosecond (fs) laser operating at 1028 nm. Silver-based refere...

A new diagnostic approach for assessing the in-depth laser induced modifications upon ultraviolet polymer irradiation is presented. The methodology relies on the observation of morphological alterations in the bulk material (Paraloid B72) by using third harmonic generation. This non destructive methodology allows the detailed and accurate imaging o...

Femtosecond laser assisted nanosurgery of microscopic biological specimens is a relatively new technique which allows the selective disruption of sub-cellular structures without causing any undesirable damage to the surrounding regions. The targeted structures have to be stained in order to be clearly visualized for the nanosurgery procedure. Howev...

Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harm...

Long term in vivo observations at large penetration depths and minimum sample disturbance are some of the key factors that have enabled the study of different cellular and tissue mechanisms. The continuous optimization of these aspects is the main driving force for the development of advanced microscopy techniques such as those based on nonlinear e...

We present a portable ultrafast Semiconductor Disk Laser (SDL) (or vertical extended cavity surface emitting laser—VECSELs), to be used for nonlinear microscopy. The SDL is modelocked using a quantum-dot semiconductor saturable absorber mirror (SESAM), delivering an average output power of 287 mW, with 1.5 ps pulses at 500 MHz and a central wavelen...

In this study, we demonstrate the potential of employing third harmonic generation (THG) imaging microscopy measurements for cell tracking studies in live Caenorhabditis elegans (C. elegans) embryos. A 1028-nm femtosecond laser was used for the excitation of unstained C. elegans samples. Different C. elegans embryonic stages (from two-cell to three...

Ultrashort pulsed laser systems (such as Ti:sapphire) have been used in nonlinear microscopy during the last years. However, its implementation is not straight forward as they are maintenance-intensive, bulky and expensive. These limitations have prevented their wide-spread use for nonlinear imaging, especially in "real-life" biomedical application...

We present the use of novel nonlinear imaging, terahertz time-domain spectroscopy and imaging as powerful diagnostic tools for studies of works of art. It is shown that nonlinear imaging offers precise in-depth information, while terahertz imaging can reveal hidden objects and uncover information on highly absorbing organic compounds whose visualiz...

To fully exploit the potential of nonlinear (NL) microscopy and enable its integration in clinical studies, inexpensive, simple, compact, efficient and sample-compatible ultrafast laser systems must be designed. Based on this, sample damage and NL signal enhancement criteria [1]; we propose the use of a compact (140x240x70 mm) semiconductor disk la...

The non-destructive determination of layer structures in works of art remains a significant challenge. Non-linear microscopy and confocal Raman microscopy (CRM) were employed for characterisation of varnish-media layers in model samples, providing important information regarding the thickness of materials and the identification of different media i...

The cleaning of over-paint layers is a usual requirement to be met in the conservation of modern paintings. In most cases, acrylics or other related compounds must be removed without affecting the original painting. The elucidation of the extent of any photochemical or structural modification induced to the substrate following laser irradiation is...

C. elegans embryogenesis, at the cell division stage, was imaged using third harmonic generation microscopy employing ultrashort pulsed lasers at 1028nm and 1550nm. This technique could be used for cell tracking studies without fluorescent markers.

In this study, third-harmonic generation (THG) imaging measurements were performed to characterize different developmental stages of the nematode Caenorhabditis elegans (C. elegans) embryos. Femtosecond laser pulses (1028 nm) were utilized for excitation. THG image contrast modality proved as a powerful diagnostic tool, providing valuable informati...

In this study, we present the implementation of non-linear spot measurements for obtaining specific and novel information related to various types of natural and synthetic glues used for lining of painted artworks. Third harmonic generation measurements were employed, in transmission mode, for the accurate and non-destructive thickness detection of...

In this study Second and Third harmonic generation (SHG-THG) imaging measurements were performed to the pharyngeal muscles of the nematode Caenorhabditis elegans, in vivo with linearly polarized laser beam. Complementary information about the anatomy of the pharynx and the morphology of the anterior part of the worm were extracted. THG signals prov...

A critical challenge for the broader implementation of laser cleaning interventions in modern paintings is the assessment of the extent of any photochemical or structural modifications induced in the original substrate following laser irradiation. For this reason, we investigate several polymeric materials (PMMA, Paraloid B72) doped with aromatic p...

We present the detailed imaging of structures and processes of the nematode Caenorhabditis elegans (C. elegans) using non-linear microscopy. Complementary information about the anatomy of the nematode was collected by implementing a combination of two photon excitation fluorescence (TPEF), second and third harmonic generation (SHG and THG) image co...

We present an appropriate methodology and results for using third-harmonic generation (THG) modality for nondestructive high resolution imaging measurements of varnished structures in model painted artifacts. Detection takes place in the reflection mode, demonstrating the ability of the technique to be applied to the evaluation of original artworks...

In this study, neurodegeneration phenomena were investigated, by performing third harmonic generation imaging measurements on the nematode Caenorhabditis elegans, in vivo. The in vivo, precise identification of the contour of the degenerating neurons in the posterior part of the nematode and the monitoring, in real time, of the progression of degen...

We present the detailed imaging of structures and processes of the nematode Caenorhabditis elegans (C. elegans) using non-linear microscopy (TPEF-SHG-THG). Consummative and specific information about the anatomy of the nematode was collected by implementing a combination of THG, SHG and TPEF image contrast modalities on the same microscope. Additio...

We present results of the implementation of three-photon excitation fluorescence (3PEF) and third harmonic generation imaging measurements for the precise and nondestructive detection of natural and synthetic varnish layers, which are used for the surface protection of painted artifacts. For this purpose, we employ as an excitation source a compact...

In this study, we use combined two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and third-harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtose...

Materials processing by ultrafast lasers offers several attractive possibilities for micro/nano materials processing. Prospects arising will be discussed in the context of surface and in bulk laser induced modifications. In particular, examples of diverse applications including the development and functionalization of laser engineered surfaces, the...

A new method is demonstrated, where three-dimensional protein structures are made by employing multi-photon polymerization and photobiotin photolysis. The technique enables the construction of arbitrary two- and three-dimensional shapes with submicron resolution. The integrity of the immobilized biotin is confirmed by derivatization with fluorescen...

In this study, we present the detailed imaging of the nematode Caenorhabditis elegans (C. elegans) at microscopic level by performing Two-Photon Excitation Fluorescence (TPEF), Second-Harmonic Generation (SHG) and Third Harmonic Generation (THG) measurements. Due to their inherent advantages in comparison with the conventional microscopy (increased...

We have demonstrated micromachining of bulk 3C silicon carbide (3C- SiC) wafers by employing 1028nm wavelength femtosecond laser pulses of energy less than 10 nJ directly from a femtosecond laser oscillator, thus eliminating the need for an amplified system and increasing the micromachining speed by more than four orders of magnitude.

Materials processing by ultrafast lasers offers several distinct possibilities for micro/nano scale applications. This is due to the unique characteristics of the laser-matter interactions involved, when sub-picosecond pulses are employed. Prospects arising will be discussed in the context of surface and in bulk laser induced modifications. In part...

In the last three years we have prepared and studied the polar methanolic extract PMF, of the herb Hypericum perforatum L, and studied as a new, alternative photosensitizing substance for PDT. Hypericum perforatum L., as well as PMF, contains a number of naphthodianthrone derivatives (hypericins), such as hypericin and pseudohypericin, as its main...

The authors demonstrate here a method for three-dimensional patterning of proteins and other biological molecules. The method employs femtosecond-laser-induced three-photon polymerization, a technique which enables the construction of arbitrary two- and three-dimensional structures of submicron resolution. Biotin is subsequently attached to the thr...

We report two-photon polymerization of an acrylate composite using a femtosecond laser at 1028 nm. The composite consists of an acrylate monomer, a free-radical co-initiator and a photo-sensitizer. The material is transparent to infrared laser radiation and absorbs strongly in the visible. By scanning the tightly focused laser beam, we employ two-p...

We demonstrate the enhancement of the wetting properties of smart photochromic surfaces by a specifically developed, gentle interferometric patterning employing green light. We realized photochromic gratings with 2.5–10.0 μ m period by a blend consisting of a green-curable matrix and spiropyran molecules. The structured surfaces exhibit photocontro...

Collagen is known to be a very efficient producer of both second harmonic generation (SHG) and two-photon excited fluorescence and the combined use of those nonlinear signals is emerging as a new imaging probe to be used as a diagnostic tool. By recording structural information of collagen between different samples, the technique shows promising fo...

We report the preparation of the methanolic extract (ME), and polar methanolic fraction (PMF) from the plant Hypericum perforatum L. The extracts contain various photosensitizing constituents such as naphthodianthrone derivatives (in 1.37% w/w), and chlorophylls (in 0.08% w/w). Upon light emission these constituents can be activated, providing phot...

We report the fabrication of three-dimensional structures of submicrometer resolution by three-photon polymerization. This resolution has been achieved by polymerizing ORMOCER, a UV photocurable organic–inorganic hybrid material, with an ultrafast laser irradiation at 1028 nm . To our knowledge, this is the first demonstration of three-photon polym...

In the current paper, a study is made of the manufacture of three-dimensional structures of micron resolution using two- and three-photon polymerization. A femtosecond laser operating at a wavelength of 1028 nm is employed for the multi-photon polymerization processes. For the component manufacture, two different materials are used. The first mater...

Autologous bone marrow transplantation is a therapeutic modality that increases the survival rates for children with malignancies with poor prognosis but relapse rates are high and attributed partially to the existence of residual malignant cells. Photodynamic treatment (PDT) has been developed among purging strategies. We investigated the effect o...

We demonstrate surface micromachining of bulk 3C silicon carbide (3C-SiC) wafers by employing tightly focused infrared femtosecond laser pulses of energy less than 10 nJ directly from a femtosecond laser oscillator, thus eliminating the need for an amplified system and increasing the micromachining speed by more than four orders of magnitude. In ad...

Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structur...

One of the main goals in optical characterization of biopsies is to discern between tissue types. Usually, the theory used for deriving the optical properties of such highly scattering media is based on the diffusion approximation. However, biopsies are usually small in size compared to the transport mean free path and thus cannot be treated with s...

We are reporting elsewhere, the promising photodynamic effect of Hypericum perforatum L. extract (PMF) against T24, NBT-II tumor bladder cells, and HL-60 leukemic cells (using 630nm, and 530nm laser light respectively). The main advantages of the extract as a photosensitizer are its low cost, extensive availability, adequate solubility, minimal tox...

Second-harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are relatively new and promising tools for the detailed imaging of biological samples and processes at the microscopic level. By exploiting these nonlinear phenomena phototoxicity and photobleaching effects on the specimens are reduced dramatically. The main target of th...

Diffusion theory is currently used to characterize highly scattering media from time and/or spatially resolved measurements. However, many of the limits of validity of the diffusion approximation in its standard form have been set through assumptions taken during its derivation, and it is generally believed that the diffusion equation holds only fo...

Laser-induced fluorescence spectra were obtained during the exposure of lamb heart (n=20) tissue to Argon-ion radiation (457.9nm). Fluorescence spectra from different heart compartments (the left and right atria and ventricles, the myocardium, the epicardium, and the aorta) were recorded. Simple algebraic algorithms based on the spectral intensity...

Dynamic scattering of coherent light by moving particles causes a Doppler shift of the original frequency, depending on the velocity and the scattering angles. This phenomenon was used for the detection of abnormal spontaneous muscle activity caused by the denervation of muscles. Transmission measurements of low frequency modulated laser irradiatio...

Optical characterization of biopsies can be used to discern between tissues when performing diffuse optical tomography of the female breast. The theory used for deriving the optical properties of such highly scattering media is based on the diffusion approximation. However, focusing the study in the regime of geometries comparable to the scattering...

Laser-induced fluorescence spectra were recorded during the exposure of lamb heat (n = 20) tissue to Argon-ion radiation (457.9 nm). Spectra from the left and right atria and ventricles, the myocardium, the epicardium, and the aorta were recorded. Simple algorithms based on the spectral intensity variation were developed in order to detect spectral...