KEYWORDS: In vivo imaging, Tissues, Windows, Animal model studies, Nonlinear optics, Microlens, Optical microscopy, Histopathology, Tissue optics, Signal generators
Tissue histopathology, reliant on costly and time-consuming hematoxylin and eosin (H&E) staining of thin tissue slices, faces limitations. Label-free non-linear optical microscopy in vivo presents a solution, allowing work on fresh samples. Implantable microstructures prove effective for systematic longitudinal in vivo studies of immunological responses to biomaterials using label-free non-linear optical microscopy. Employing two-photon laser polymerization, we implanted a matrix of 3D lattices in the chorioallantoic membrane of chicken embryos, establishing a 3D reference frame for cell counting. H&E analysis is compared to label-free in vivo non-linear excitation imaging for cell quantification and identifying granulocytes, collagen, and microvessels. Preliminary results in higher animal models demonstrate the transformative potential of this approach, offering an alternative to conventional histopathology for validating biomaterials in in vivo longitudinal studies.
Deep tissue imaging remains challenging, especially for thick media, due to spherical aberrations induced on focused beams by the tissue. In this framework we propose a miniaturized in-vivo imaging window composed of high dioptric power microlenses coupled to micro-scaffold, which were fabricated by two-photon polymerization (2PP) in the biocompatible photoresist SZ2080. We adopt a single-irradiation strategy for the fabrication of the whole structure: We first irradiate the micro-scaffold on the bottom side of the chip and afterwards the microlenses on the top. For the microlenses we adopted a hybrid approach by combining the 2PP of the micro lenses surface with a subsequent UV crosslinking of the inner volume. We explored different lenses profiles (plano-convex and parabolic) with variable parameters like diameter and focal lengths, to optimize the imaging characteristics. We envisage that these imaging windows will open the way to direct and continuous optical inspection of biological processes in vivo.
In the context of biomaterials, small-molecules and drugs testing, intravital microscopy allows to quantify in-vivo the immune reaction, reducing the number of laboratory animals required to statistically validate the product. However, fluorescence microscopy is affected by limited tissue penetration due to light scattering and by optical aberrations, induced on focused beams, by the animal tissue surrounding the implant. In this framework, we developed a system of microlenses coupled to microscaffolds, both incorporated in a miniaturized imaging window. The chip is designed to act as an in-situ microscope objective with the aim to overcome the restrictions of in-vivo imaging (i.e. spherical aberrations) and to allow multiple biological observations in the same animal (by including fluorescent beacons). The device is fabricated by two-photon polymerizing a biocompatible photoresist called SZ2080. The microlenses are manufactured by the concentric polar scanning of the laser beam to realize their outer surface, followed by the UV bulk polymerization of their inner SZ2080. We preliminarily characterized the imaging capabilities of our implantable system on live cells cultured on flat substrates and 3D microscaffolds by coupling it to low magnification objectives. The microlenses optical quality is sufficient to induce non-linear excitation and collect two-photon excitation images with the same level of laser intensity and signal-to-noise ratio. Remarkably, they allow to efficiently excite the fluorescence of labelled human fibroblasts collecting high resolution magnified images. These results will open the way to the application of implanted micro-optics for the real-time and continuous in-vivo observation of complex biological processes.
Despite their key-role during the histopathological diagnosis, staining procedures are expensive and time-consuming. Label-free microscopy provides an alternative since it allows the visualization of endogenous proteins without the need of extrinsic dyes. SuperµMAPPS, a novel AI-based method, analyzes the Polarized Second Harmonic Generation signal from collagen to characterize its micro-architecture in terms of fibrils mean orientation θF and anisotropy γ, related to tumor development. After a proper validation on synthetic images, human breast cancer samples at different growth stages have been analyzed through SuperµMAPPS, highlighting its capability to detect tumorous tissue at early stages in a real clinical context.
We present a new AI-based method for the quantification of liver fibrosis in tissue sections stained with Picro Sirius Red which highlights collagen. The method segments and quantifies collagen, a marker of the fibrotic response, through a deep learning model trained on 20 whole-slide images. The results show a Dice score > 90% compared to manual annotations, demonstrating its potential aid during diagnosis. Furthermore, our approach can be extended to other staining protocols.
H&E stained sections are the gold standard for disease diagnosis but, unfortunately, the staining process is time-consuming and expensive. In an effort to overcome these problems, here, we propose a virtual staining algorithm, able to predict an Hematoxylin/Eosin (H&E) image, usually exploited during clinical evaluations, starting from the autofluorescence signal of entire liver tissue sections acquired by a confocal microscope. The color and texture contents of the generated virtually stained images have been analyzed through the phasor-based approach to detect tumorous tissue and to segment relevant biological structures (accuracy>90% compared to the expert manual analysis).
We report new methods of two-photon polymerization of microlenses with high numerical aperture, large diameter and good optical quality. We characterize the aberrations of these lenses that, coupled to raster scanning optical microscopes, allow two-photon excitation imaging of cells. In-vivo non-linear imaging will be also discussed, opening the possibility to use these micro-lenses in implants for the continuous inspection of biological dynamics in vivo.
The current protocols for biocompatibility assessment of biomaterials, based on histopathology, require the sacrifice of a huge number of laboratory animals with an unsustainable ethical burden and remarkable cost. Intravital microscopy techniques can be used to study implantation outcomes in real time though with limited capabilities of quantification in longitudinal studies, mainly restricted by the light penetration and the spatial resolution in deep tissues. We present the outline and first tests of a novel chip which aims to enable longitudinal studies of the reaction to the biomaterial implant. The chip is composed of a regular reference microstructure fabricated via two-photon polymerization in the SZ2080 resist. The geometrical design and the planar raster spacing largely determine the mechanical and spectroscopic features of the microstructures. The development, in-vitro characterization and in vivo validation of the Microatlas is performed in living chicken embryos by fluorescence microscopy 3 and 4 days after the implant; the quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration.
I will review the fundamental limiting issues in in-vivo optical imaging and discuss the proposal made by the IN2SIGHT consortium to overcome them and its impact. IN2SIGHT will foster a breakthrough in in-vivo optical imaging that will renovate the biocompatibility tests (ISO10993 EU norm) required for the development of biomaterials for clinical use. These tests are economical and ethical unsustainable for small-medium industries and for the society.
The IN2SIGHT approach stands on a micro-structured chip that will recast our thinking of deep tissue in-vivo imaging.
I discuss how this proposal will allow unique quantification of the immune reaction to biomaterials, thus reducing time and costs for testing with a potential huge impact on public health systems and our society. The project sees the collaboration of seven partners from five countries and will exploit from the beginning inter-sectorial approaches in an interdisciplinary environment.
We describe several bioengineered cell models developed by our group. We develop tools for cell culture allowing fluorescence diagnostics on the cellularised constructs cultured within, both in 3D and prolonged culture times extending to several weeks. These cell models proved able to recapitulate in vitro several slowly developing biological processes such as the regeneration of a cartilaginous tissue by cartilage cells, the formation of a bone metastasis by breast cancer cells, the instruction of adaptive immune cells as occurs in a lymphnode, and the neuroprotective effect on pathological neurons of mesenchymal stem cell secretome. We also scaled down these tools in the aim to better control stem cell function in our models, by applying two-photon laser polymerization to fabricate micro scaffolds for cell expansion. We were able to condition mesenchymal stem cells, neural precursor cells and embryonic stem cells towards maintenance of a greater stemness and multipotency or pluripotency, compared to conventional flat culture. This result opens up an avenue towards a safer use of these cells for stem cells therapies. Finally, we describe a revolutionary concept to implant the cell model in a living organism, to be revascularized by the host allowing for studies involving interactions with the host immune system.
We exploit two-photon laser writing to fabricate 3D biocompatible proteinaceous microstructures (∼1 to 50 𝜇m in lateral size) with tunable elasticity and photo-thermal activity in the near-infrared. Structure printing relies on the photo-crosslinking of the protein bovine serum albumin (BSA, 50 mg/mL) initiated by the Rose Bengal dye (2 mM concentration), whereas photo-thermal functionality is achieved by the dispersion of non-spherically symmetric metallic nanoparticles into the ink.
Aiming at a subsequent application of the fabricated microstructures as platforms for cell growth and stimulation, we carry out a thorough characterization of their mechanical and photo-thermal properties. Preliminary data obtained by AFM indentation have quantified the structures Young modulus in the broad 100-1000 kPa range depending on the BSA concentration. Stiffness is further characterized here by subjecting the fabricated microstructures to steady flow in a microfluidic device, and by quantifying their real-time bending by a conventional transmitted light microscope. In parallel, we focus on the optimization of the photo-thermal activity of the structures. Anisotropic gold nanoparticles, dispersed in the ink, get trapped into the structure during photo-crosslinking and lead to localized heat release upon excitation in the near-infrared. The temperature increment is rapidly (∼1 s) reached and maintained under continuous wave laser irradiation at 800 nm; the amplitude of the temperature variation is quantified as a function of the incident laser power by means of infrared thermography and is correlated to both the structure thickness and the nanoparticles concentration. The resulting spatially confined heat loads could be exploited to induce highly localized responses in cells. In this direction, proteinaceous photo-thermal microstructures can be used to physically induce the differentiation of cells (e.g. neurons or fibroblasts) in a spatially controlled manner.
Mapping flows in vivo is essential for the investigation of cardiovascular pathologies in animal models. The limitation of optical-based methods, such as space-time cross correlation, is the scattering of light by the connective and fat components and the direct wave front distortion by large inhomogeneities in the tissue. Nonlinear excitation of the sample fluorescence helps us by reducing light scattering in excitation. However, there is still a limitation on the signal-background due to the wave front distortion. We develop a diffractive optical microscope based on a single spatial light modulator (SLM) with no movable parts. We combine the correction of wave front distortions to the cross-correlation analysis of the flow dynamics. We use the SLM to shine arbitrary patterns of spots on the sample, to correct their optical aberrations, to shift the aberration corrected spot array on the sample for the collection of fluorescence images, and to measure flow velocities from the cross-correlation functions computed between couples of spots. The setup and the algorithms are tested on various microfluidic devices. By applying the adaptive optics correction algorithm, it is possible to increase up to 5 times the signal-to-background ratio and to reduce approximately of the same ratio the uncertainty of the flow speed measurement. By working on grids of spots, we can correct different aberrations in different portions of the field of view, a feature that allows for anisoplanatic aberrations correction. Finally, being more efficient in the excitation, we increase the accuracy of the speed measurement by employing a larger number of spots in the grid despite the fact that the two-photon excitation efficiency scales as the fourth power of this number: we achieve a twofold decrease of the uncertainty and a threefold increase of the accuracy in the evaluation of the flow speed.
Ramification of blood circulation is relevant in a number of physiological and pathological conditions. The oxygen exchange occurs largely in the capillary bed, and the cancer progression is closely linked to the angiogenesis around the tumor mass. Optical microscopy has made impressive improvements in in vivo imaging and dynamic studies based on correlation analysis of time stacks of images. Here, we develop and test advanced methods that allow mapping the flow fields in branched vessel networks at the resolution of 10 to 20 μm. The methods, based on the application of spatiotemporal image correlation spectroscopy and its extension to cross-correlation analysis, are applied here to the case of early stage embryos of zebrafish.
Optical Microscopy has been applied to life science from its birth and reached widespread application due to its major advantages: limited perturbation of the biological tissue and the easy accessibility of the light sources. However, as the spatial and time resolution requirements and the time stability of the microscopes increase, researchers are struggling against some of its limitations: limited transparency and the refractivity of the living tissue to light and the field perturbations induced by the path in the tissue. We have developed a compact stand-alone, completely scan-less, optical setup that allows to acquire non-linear excitation images and to measure the sample dynamics simultaneously on an ensemble of arbitrary chosen regions of interests. The image is obtained by shining a square array of spots on the sample obtained by a spatial light modulator and by shifting it (10 ms refresh time) on the sample. The final image is computed from the superposition of (100-1000) images. Filtering procedures can be applied to the raw images of the excitation array before building the image. We discuss results that show how this setup can be used for the correction of wave front aberrations induced by turbid samples (such as living tissues) and for the computation of space-time cross-correlations in complex networks.
The research and development of personalized medical treatments is increasing steadily fostered by its large societal impact. The ability of non-spherical gold nanoparticles to locally and efficiently release heat when irradiated in Near Infrared (NIR) wavelength region is a promising tool for photothermal medical therapies. In the present work, stable inks containing PEGylated gold nanostars (GNS) were obtained and inkjet-printed on a pigment coated paper substrate. Significant photothermal effect of the printed patterns was observed under Near Infrared (NIR) excitation of the Localized Surface Plasmon Resonance (LSPR) of the GNS. These preliminary results support, in perspective, the application of printed GNS patterns for thermal medical treatments either by direct localized heating, or by temperature triggered drug release.
Gold nanocages (AuNCs) have been shown to be a useful tool both for imaging and hyperthermia therapy of cancer, thanks to their outstanding optical properties, low toxicity and facile functionalization with targeting molecules, including peptides and antibodies. In particular, hyperthermia is a minimally invasive therapy which takes advantage of the peculiar properties of gold nanoparticles to efficiently convert the absorbed light into heat. Here, we use AuNCs for the selective targeting and imaging of prostate cancer cells. Moreover, we report the hyperthermic effect characterization of the AuNCs both in solution and internalized in cells. Prostate cancer cells were irradiated at different exposure times, with a pulsed near infrared laser, and the cellular viability was evaluated by confocal microscopy.
Microcirculation plays a key role in the maintenance and hemodynamics of tissues and organs also due to its extensive interaction with the immune system. A critical limitation of state-of-the-art clinical techniques to characterize the blood flow is their lack of the spatial resolution required to scale down to individual capillaries. On the other hand the study of the blood flow through auto- or cross-correlation methods fail to correlate the flow speed values with the morphological details required to describe an intricate network of capillaries. Here we propose to use a newly developed technique (FLICS, FLow Image Correlation Spectroscopy) that, by employing a single raster-scanned xy-image acquired in vivo by confocal or multi-photon excitation fluorescence microscopy, allows the quantitative measurement of the blood flow velocity in the whole vessel pattern within the field of view, while simultaneously maintaining the morphological information on the immobile structures of the explored circulatory system. Fluorescent flowing objects produce diagonal lines in the raster-scanned image superimposed to static morphological details. The flow velocity is obtained by computing the Cross Correlation Function (CCF) of the intensity fluctuations detected in pairs of columns of the image. The whole analytical dependence of the CCFs on the flow speed amplitude and the flow direction has been reported recently. We report here the derivation of approximated analytical relations that allows to use the CCF peak lag time and the corresponding CCF value, to directly estimate the flow speed amplitude and the flow direction. The validation has been performed on Zebrafish embryos for which the flow direction was changed systematically by rotating the embryos on the microscope stage. The results indicate that also from the CCF peak lag time it is possible to recover the flow speed amplitude within 13% of uncertainty (overestimation) in a wide range of angles between the flow and the image scanning direction.
We have previously addressed experimentally blood fluidodynamics in microcapillaries by coupling optical microscopy to pixelated detection. By computing the Cross-Correlation Function (CCF) of signals coming from pixels at a distance along the flow we obtained information on the flow speed and direction. The extension of these experiments to more complex systems with high branching of capillaries and/or inverted flows needs a theoretical investigation that we present here. We focus first on straight capillaries and harmonic flows between a minimum Vmin ≠ 0 and a maximum Vmax flow speed. The CCF shows multiple peaks at lag times that correspond closely to the maximum and minimum flow speeds. The general analytical expression of the CCF is given, the position of its maxima are discussed by means of geometrical considerations and numerical analysis and an experimental validation are presented. The second case that we study is the flow in the branches of a y-shaped junction in a microcapillary. By simply modeling the branching in laminar flow (low Reynold numbers) and assuming a smooth transition of speeds along the branches we derive a simple numerical model to compute the trajectories of micro-beads. We estimate the flow speed in the branches by computing the CCFs between linear regions of interest set perpendicular to the axes of the branches.
The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup (SLM-2PM) which collects three-dimensional two-photon images by only exploiting the SLM. This configuration leads to an accurate placement of laser beamlets over small focal volumes, eliminating mechanically moving parts and making the system stable over long acquisition times. Fluorescence signals are diffraction limited and are acquired through a pixelated detector, setting the actual limit to the acquisition rate. High-resolution structural images were acquired by raster-scanning the sample with a regular grid of excitation focal volumes. These images allowed the selection of the structures to be further investigated through an interactive operator-guided selection process. Functional signals were collected by illuminating all the preselected points with a single hologram. This process is exemplified for high-speed (up to 1 kHz) two-photon calcium imaging on acute cerebellar slices.
Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern. To overcome this limitation, we develop here a two-photon two-spots setup coupled to an all-reflective near-infrared (NIR) optimized scanning system and to an electron multiplying charge-coupled device. Two NIR laser spots are spaced at adjustable micron-size distances (1 to 50 μm) by means of a Twyman-Green interferometer and repeatedly scanned on the sample, allowing acquisition of information on flows at 4 ms–3 μm time-space resolution in parallel on an extended field of view. We analyze the effect of nonhomogeneous and variable flow on the cross-correlation function by numerical simulations and show exemplary application of this setup in studies of blood flow in zebrafish embryos in vivo. By coupling the interferometer with the scanning mirrors and by computing the cross-correlation function of fluorescent red blood cells, we are able to map speed patterns in embryos’ vessels.
The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones.
P53 is a tumor suppressor used as marker for early cancer diagnosis and prognosis. We have studied constructs based on
gold nanoparticles (NPs) decorated with specific anti-p53 antibodies and with a fluoresceine derivative, FITC. The
interaction of gold surface plasmons with fluorophores bound within few nanometers from the surface, likely induces
changes in the fluorophore excited state lifetime. Indeed we found previously that this parameter follows linearly the p53
concentration in solutions (in vitro conditions) up to 200-400 pM, depending on the size of the NP, with a 5 pM
uncertainty. We have evaluated here the nanosensor specificity for p53 by testing it in-vitro against bovine serum
albumine, beta-lactolglobulin and lysozyme. Moreover, the titration of total cell extracts from p53+/+ or p53-/- cells with
the p53antibody decorated gold NPs, indicates that this construct can also be used to detect the presence of p53 in total
cell extracts and it will be therefore a valuable tool also for in vivo screening.
KEYWORDS: Diffusion, Tissues, Two photon excitation microscopy, Pathogens, Microscopes, In vivo imaging, Signal detection, Physics, Microscopy, Inspection
Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells
(NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen
associated molecular pattern (PAMP) activated-DCs in inducing NK
cell-mediated anti-tumor responses.
Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected
with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4
dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory
conditions are also evaluated.
Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar
kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK
dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and
average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might
efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK
cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs.
We describe and apply two raster scanning algorithms that
are suited for fast imaging and for spectroscopy in a two
photon microscope. Imaging can be performed at a rate
of 1-100 Hz per line with a closed loop piezo-actuator.
In order to reach the single molecule sensitivity and to
study the dynamics of the fluorescence emission, the
detection is performed via avalanche photodiodes.
In a slow scanning algorithm we have implemented
photon counting histogram and lifetime analysis on
the image. In this way we are able to discriminate
between local concentration and molecular brightness
and to measure lifetimes on extended samples.
The one photon and two photon excitation spectral properties (absorption, emission spectra, singlet lifetime) of a very efficient two photon absorber, dimethyl-pepep, have been measured in solution. The one photon excitation peak lye near 525 nm and the emission falls at 600 nm, where autofluorescence of cells is weak. The value of the singlet-triplet conversion rate, obtained by two-photon excitation fluorescence correlation spectroscopy, has a quadratic dependence on the excitation power and is comparable to that shown by the dye rhodamine. Preliminary results on stained cells from yeast Saccaromices cerevisiae and Paramecium primaurelia show that the dye preferentially stains DNA in the cell. A direct comparison with a DNA stainer, Dapi, is also performed. Some measurements of the dye functionalized to react with lysine and n-terminal residues of protein are presented. Moreover, this dye can be employed in order to follow in detail some cellular processes such as nuclei division. In vitro fluorescence titration of dimethyl-pepep with calf thymus DNA allowed to estimate the values of the dye-DNA association constant versus ionic strength, and an affinity close to that of ethidium bromide is found.
Confocal microscopy is one of the most widely used and non-invasive tool for the investigation of biological matter. It improves the performances of the optical microscope by reducing the excitation volume and enhancing the axial resolution due to the use of high numerical aperture lenses. We have adapted an inverted confocal optical microscope to the measurement of fluorescence emission dynamics (lifetime and fluorescence polarization anisotropy). The dynamic spectroscopy measurements are obtained with phase fluorometry and are based on a modulated linearly polarized laser beam which is fed to the epifluorescence port of the microscope. We report the
test of the microscope by comparing the lifetime and fluorescence polarization anisotropy decay obtained in cuvettes in the standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor is introduced in the best fit functions used in the data analysis of the decays, the results obtained on microliters volumes is comparable to those obtained in cuvettes in the standard phase fluorometer spectrometer. An example of application is reported on short DNA fragments.
The synthesis and characterization of novel heteroaromatic-based two-photon absorption (TPA) dyes for bio-conjugation is described; the new isothiocyanate and maleimide derive from a class of novel efficient quadrupolar and octupolar/branched chromophores relying on the electronic effects of electron-poor and electron-rich simple heteroaromatic rings; the new systems exhibit very large TPA cross-sections, high chemical stability, and very low photobleaching.
Single molecule spectroscopy often requires the immobilization of the molecules onto solid or quasi-solid substrates and the use of relatively high excitation intensity. We have studied the fluorescence emission of four common dyes used for bio-imaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level under two-photon excitation regime. We focus on two-photon excitation thermal effects on the stability of the single molecules, influencing the internal photo-dynamics and the total duration of the fluorescent emission. Single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output till a sudden transition to a dark state. The bleaching time varies in the series pyrene, indo-1, fluorescein and rhodamine 6g from the fastest to the slowest one respectively, has a gaussian distribution suggesting that bleaching is not due to photo-bleaching. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon excitation process and the thermal bleaching is correlated to the amount of absorbed power that is not re-irradiated as fluorescence.
Three-dimensional confocal laser scanning microscopy (CLSM) and two-photon excitation microscopy (TPEM) were used to study the response of cellular systems to fuzzy organized nanostructured polyelectrolytes used both as microcontainers and microcarriers for drug delivery. These nanostructured systems are named Nanocapsules and represent a new class of controllable colloids. CLSM and TPEM uniquely allow to follow the fate of encapsulated living cells and to track the pathway of nanocapsules introduced into cellular systems. For the former situation, it will be shown how living cells can be encapsulated and demonstrated the preservation of the metabolic and duplicating activity. In this case the role of the Nanocapsule is as microcontainer endowed of functionalized surface and of protective ability. The latter situation, is related to feeding living cells with Nanocapsules. This experiment serves in elucidating the comprehension of the potential cytotoxicity and of the ability of Nanocapsules to reach specific targets where active compounds can be released. Cellular systems used within this research are Saccharomyces cerevisiae and Paramecium primaurelia living cells. In the case of encapsulation of Saccharomyces cerevisiae living cells, the most relevant result is that, after encapsulation, cells preserve their metabolic activities and they are still able to divide. At this stage is also relevant the utilization of spectroscopic methods like fluorescence lifetime and second harmonic imaging. These hybrid polyelectrolyte-cells can provide a cheap model system in a wide range of biophysical and biotechnological applications, thanks to the tunable properties of the polyelectrolyte shell.
Confocal and Two-photon excitation laser scanning microscopy allow gathering three-dimensional and temporal information from biological systems exploiting fluorescence labeling and autofluorescence properties. In this work we study biological events linked to functionality in Paramecium primaurelia. The internalization of material in ciliated one-celled organisms (protozoa) occurs via different mechanisms, even if most of nutrients, particulate or not, is taken up by food vacuoles formed at the bottom of the oral cavity. The endocytosis of small-sized molecules occurs at the parasomal sacs, located next the ciliar basal bodies. Vital fluorescent dyes (BSA-FITC, WGA-FITC, dextran-Texas Red, cholesteryl-Bodipy) and autofluorescence were used to study formation, movement, and fusion of vesicles during endocytosis and phagocytosis of Paramecium primaurelia. By immobilizing living cells pulsed with food vacuole and endosome markers at successive times after chasing in unlabeled medium, the intracellular movement and fusion of food vacuoles and of endosomes were visualized. A temporal analysis of fluorescence images and the false-color technique were used. Starting from time series or 3D data sets composite images were generated by associating with each originally acquired image a different color corresponding to each sampling point in time and along the z-axis. Second Harmonic Generation Imaging attempts are also outlined.
We have combined a confocal laser scanning head modified for TPE with some spectroscopic modules to study single molecules and molecular aggregates of rhodamine 6G and labeled proteins on glass substrates. The fluorescence intensity of the spots occurs at definite values that are multiples of a reference signal and extrapolate to the background level measured on the images. These properties suggest that these spots arise from single rhodamines. The discrete character of the intensity distribution can therefore be used as a simple, quantitative tool to discriminate between single molecules and molecular aggregates on single snapshots. These studies have been performed by using a combined confocal and spectroscopic architecture realized for two-photon excitation.
The effect of the insertion of different amounts (from 0 to 6) of the curved sequence AluI in pUC18m plasmid (2686 base pairs, bp) is studied by dynamic light scattering. This sequence is a highly repeated 113 base pairs long sequence from Artemia Franciscana shrimp. A 30% compaction of the plasmids containing 2 and 6 adjacent AluI sequences compared to pUC8 plasmid (2717 bp) is observed. Furthermore the behavior of the translational diffusion coefficient Dt versus the number of adjacent AluI insertion is not monotonic.
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