We will present the multi-modal photonic platform including Optical Tweezers, linear and non linear optics techniques in a single instrument to allow parallel information gathering during single cell processes. The platform includes the following techniques: multipoint Optical Tweezers; Laser cutting; multi/single photon fluorescence, Fluorescence Lifetime Imaging (FLIM); Förster Resonant Energy Transfer (FLIM-FRET); Fluorescence Correlation Spectroscopy (FCS); Raman; Second/Third Harmonic Generation (SHG/THG); Coherent AntiStokes Raman Scattering (CARS) and cascade CARS; Near field tip-enhancement and 1 and 2 photons Photoluminescence Excitation Spectroscopy (1-2 PLE). Next, we will discuss the issue of spherical wave vectors decomposition of any optical beam in the Fourier space without any approximation solving the problem of spherical Bessel functions cancellation in both sides of the expansion. This expansion is the necessary first step to perform optical forces, as well as optical signals intensities, of scattering/absorbing particles. The limit of Rayleigh regime is easily obtained.
We report the fabrication of PbTe quantum dots grown under inert gas (Ar and He) atmosphere by
pulsed laser deposition using the second harmonic of a Q-Switched Quantel Nd:YAG laser. For
characterization, samples were prepared onto a 40Å carbon film deposited on a copper grid. The
influence of background pressure, and number of laser pulses on the size distribution of the PbTe
nanoparticles was investigated by transmission electron microscopy using a 200 kV TECNAI G2
F20 electron microscope with 0.27 nm point resolution. The size distribution was obtained by
manually outlining the particles from several dozens of low- and high-resolution TEM images. Once
digitized and saved in a proper format, the image was processed using the J-image software.
Characterizations reveal an increase of the nanoparticle size both with the amount of material
deposited (number of laser pulses) and the background pressure. Furthermore, measurements reveal
a narrower nanoparticle size distribution by increasing the number of laser pulses or by decreasing
the background pressure. HRTEM studies of the influence of different ambient gases on the
structural properties of the PbTe nanoparticles are being conducted.
We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and
Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging
platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal
approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of
TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the
transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E
staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it
important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already
existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely
changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples
demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and
quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the
potential to differentiate between cancerous and healthy ovarian tissue.
Osteogenesis Imperfecta (OI) is a genetic disorder that leads to bone fractures due to mutations in the Col1A1 or Col1A2
genes that affect the primary structure of the collagen I chain with the ultimate outcome in collagen I fibrils that are
either reduced in quantity or abnormally organized in the whole body. A quick test screening of the patients would
largely reduce the sample number to be studied by the time consuming molecular genetics techniques. For this reason an
assessment of the human skin collagen structure by Second Harmonic Generation (SHG) can be used as a screening
technique to speed up the correlation of genetics/phenotype/OI types understanding. In the present work we have used
quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization of
the OI human skin samples comparing with normal control patients. By comparing fibril collagen distribution and spatial
organization, we calculated the anisotropy and texture patterns of this structural protein. The analysis of the anisotropy
was performed by means of the two-dimensional Discrete Fourier Transform and image pattern analysis with Gray-Level
Co-occurrence Matrix (GLCM). From these results, we show that statistically different results are obtained for the
normal and disease states of OI.
In this work we proposed and built a multimodal optical setup that extends a commercially available confocal
microscope (Olympus FV300) to include nonlinear optical (NLO) microscopy and fluorescence lifetime imaging
microscopy (FLIM). The NLO microscopies included two-photon fluorescence (TPFE), Second Harmonic Generation
(SHG) and Third Harmonic Generation (THG). The whole system, including FLIM, used only one laser source
composed of an 80 MHz femtosecond laser. The commercial Ti:sapphire lasers can be tuned up to 690-1040 nm bringing
the THG signal to the 350 nm region where most microscope optics do not work. However, the third harmonic is only
generated at the sample, meaning that we only have to take care of the collection optics. To do that we used a remote
photomultiplier to acquire the THG signal at the 310-350 nm wavelength window. After performing the tests to
guarantee that we are observing actually SHG/THG signals we than used this system to acquire multimodal images of
several biological samples, from epithelial cancer to vegetables. The ability to see the collagen network together with the
cell nuclei proved to be important for cancer tissues diagnosis. Moreover, FLIM provides information about the cell
metabolism, also very important for cancer cell processes.
We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG/THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium/stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.
We used human specimens of epithelial ovarian cancer (serous type) to test the feasibility of nonlinear imaging as
complementary tools for ovarian cancer diagnosis. Classical hematoxylin-and-eosin stained sections were applied to
combining two-photon excitation fluorescence (TPEF), second (SHG), and third (THG) harmonic microscopy within the
same imaging platform. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with
Hematoxylin & Eosin (H&E) stored for a very long time and that H&E staining enhanced the THG signal. We
demonstrate using anisotropy and morphological measurements, that SHG and THG of stained optical sections allow
reproducible identification of neoplastic features such as architectural alterations of collagen fibrils at different stages of
the neoplastic transformation and cellular atypia. Taken together, these results suggest that, with our viable imaging
system, we can qualitatively and quantitatively assess endogenous optical biomarkers of the ovarian tissue with SHG and
THG microscopy. This imaging capability may prove to be highly valuable in aiding to determine structural changes at
the cellular and tissue levels, which may contribute to the development of new diagnostic techniques.
In the last few years, quantum confinement effects in semiconductor nanocrystals (quantum dots - QDs) have attracted a
significant amount of interest due to their new optical properties and also because of their potential applications in
biological systems. In this work, cadmium sulphide (CdS) nanoparticles were synthesized in aqueous medium and
passivated with Cd(OH)2. Polyphosphate ions were used in order to avoid particle aggregation. After the passivation
step, CdS/Cd(OH)2 quantum dots were coated with silica. Silica coating has been extensively investigated concerning its
properties in biocompatibilizing QDs to biological systems. Silica coated core-shell CdS/Cd(OH)2 water soluble QDs
optical properties were studied by absorption, excitation and emission spectroscopies, while their morphological
characterization was carried out by transmission electron microscopy.
KEYWORDS: Quantum dots, Toxicity, Nanoparticles, In vivo imaging, In vitro testing, Cadmium, Transmission electron microscopy, Control systems, Plasma, Nanocrystals
Many studies have been done in order to verify the possible nanotoxicity of quantum dots in some cellular
types. Protozoan pathogens as Trypanosoma cruzi, etiologic agent of Chagas1 disease is transmitted to
humans either by blood-sucking triatomine vectors, blood transfusion, organs transplantation or congenital
transmission. The study of the life cycle, biochemical, genetics, morphology and others aspects of the T. cruzi
is very important to better understand the interactions with its hosts and the disease evolution on humans.
Quantum dot, nanocrystals, highly luminescent has been used as tool for experiments in in vitro and in vivo T.
cruzi life cycle development in real time. We are now investigating the quantum dots toxicity on T. cruzi
parasite cells using analytical methods. In vitro experiments were been done in order to test the interference of
this nanoparticle on parasite development, morphology and viability (live-death). Ours previous results
demonstrated that 72 hours after parasite incubation with 200 μM of CdTe altered the development of T. cruzi
and induced cell death by necrosis in a rate of 34%. QDs labeling did not effect: (i) on parasite integrity, at
least until 7 days; (ii) parasite cell dividing and (iii) parasite motility at a concentration of 2 μM CdTe. This
fact confirms the low level of cytotoxicity of these QDs on this parasite cell. In summary our results is
showing T. cruzi QDs labeling could be used for in vivo cellular studies in Chagas disease.
Semiconductor quantum dots [QD] have shown a great number of applications from fluorescent markers to solar cell
devices. Colloidal systems have been usually obtained through chemical synthesis, that have to be devoleped for each
material. The best quality QDs have been obtained with non-aqueous solution and non-physiological pH, requiring a
posterior processing to be used in biology, for example. In contrast, the same physical synthetic method, such as laser
ablation, would be applied to any semiconductor, metallic or dielectric material. Colloidal QD can be obtained by laser
ablation of a target inside any solvent, given this method a very large flexibility. The fluorescence efficiency, however,
depend on the surface traps and stability of colloids. The usual method to avoid surface traps is to grow a cap layer to
passivate its surface and, at the same time, stabilize the colloid, sterically or electrostatically.
In this work we report a novel technique for obtain thiol capped CdTe colloidal quantum dots in one step. A
target immerse in a solution of ethanol and 3-mercaptopropyltrimethoxysilane (MPS), or thiol, was hit by a nanosecond
532 nm laser. With this assembly CdTe luminescent QDs were obtained. The colloid photoluminescence and other
optical and structural properties are studied.
In this work we used a methodology to study chemotaxis of Trypanossoma cruzi (T. Cruzi) in real time using an
Optical Tweezers system. Trapped beads were used as a force transducer for measuring forces of the same order of
magnitude as typical forces induced by flagellar motion. Optical Tweezers allowed real time measurements of the force
vectors, strength and direction, of living parasites under chemical or other kinds of gradients. This seems to be the ideal
tool to perform observations of taxis response of cells and microorganisms with high sensitivity to capture instantaneous
responses to a given stimulus. We applied this methodology to investigate the T. cruzi under distinct situations: the
parasite alone and in the presence of its insect-vector Rhodnius prolixus (R. prolixus).
In this work we report a novel technique for obtain thiol capped CdTe colloidal
quantum dots in one step. These nanoparticles are compatible for silica capping indicating their
possible use as fluorescent markers.
In this work, we propose a methodology to study microorganisms chemotaxis in real time using an Optical
Tweezers system. Optical Tweezers allowed real time measurements of the force vectors, strength and direction, of
living parasites under chemical or other kinds of gradients. This seems to be the ideal tool to perform observations of
taxis response of cells and microorganisms with high sensitivity to capture instantaneous responses to a given stimulus.
Forces involved in the movement of unicellular parasites are very small, in the femto-pico-Newton range, about the same
order of magnitude of the forces generated in an Optical Tweezers. We applied this methodology to investigate the
Leishmania amazonensis (L. amazonensis) and Trypanossoma cruzi (T. cruzi) under distinct situations.
Semiconductor colloidal quantum dots have been, for the past two decades, incorporated in a wide range of
applications from catalysis and optical sensors to biolabels. For this reason, simple, cheap and reproducible routes of
synthesis are the main goal of many research groups around the world. They seek the production of a very stable and
extremely quantum efficient nanocrystal that can afford rough changes in the external environment. Silica capping is
becoming a very common tool in the quest for a stable quantum dot, because of its strong and stable structure, this
material provides a great insulator to the nanocrystal from the outside. The nanocrystal surface is not chemically
favorable to the deposition of the bare silica shell, what demands a bifunctional molecule that provides the linkage
between the core and the shell. In this work we present a comparison between several silanization methods of thiol
capped CdSe and CdTe quantum dots, showing some simplifications of the routes and an application of the quantum dots
produced as fluorescent cell markers in acquisition of confocal microscopy images.
One of the fundamental goals in biology is to understand the interplay between biomolecules of different cells. This
happen, for example, in the first moments of the infection of a vector by a parasite that results in the adherence to the cell
walls. To observe this kind of event we used an integrated Optical Tweezers and Confocal Microscopy tool. This tool
allow us to use the Optical Tweezers to trigger the adhesion of the Trypanosoma cruzi and Trypanosoma rangeli parasite
to the intestine wall cells and salivary gland of the Rhodnius prolixus vector and to, subsequently observe the sequence
of events by confocal fluorescence microscopy under optical forces stresses. We kept the microorganism and vector cells
alive using CdSe quantum dot staining. Besides the fact that Quantum Dots are bright vital fluorescent markers, the
absence of photobleaching allow us to follow the events in time for an extended period. By zooming to the region of
interested we have been able to acquire confocal images at the 2 to 3 frames per second rate.
We present Raman-scattering results for PbTe quantum dots (QDs) in doped telluride glasses which clearly
show the confinement effects on the phonon spectra as a function of the quantum-dot size.
PbTe doped tellurite glass photonic optical fiber for non linear application were developed using rod in tube method in a
draw tower. We follow the growth kinetics of the quantum dots in the optical fiber by High Resolution Transmission
Electron Microscopy giving some results related with the growth kinetic of the same in function of time so much for
optical fiber as for the glass bulk. Absorption peak near 1500 nm as observed and it was attributed the optical resonance
due PbTe quantum dots in the core fiber.
One very important contribution of the Optical Tweezers technique is its ability to extract the missing mechanical
measurements in the world of microorganisms and cells that could be correlated to biochemical information. A
microsphere displacement is the preferential force transducer for this kind of measurement. However, the typical
conditions used in Optical Tweezers with very high numerical aperture beams and microspheres with diameters up to ten
wavelengths, requires a full vectorial description of the incident beam in partial waves with the origin of coordinate
system at the center of the microsphere and not at the focus of the beam. Using the Angular Spectrum Representation of
the incident beam and an analytical expression for integrals involving associated Legendre Polynomials, Bessel
functions and plane waves we have been able to obtain a closed expression, without any approximation, for the beam
shape coefficients of any orthogonally incident beam. The theoretical prediction of the theory agrees well with the
experimental results performed on a 3D positioned dual trap in an upright standard optical microscope, thus obtaining
the whole optical force curves as a function of the microsphere center for different wavelengths.
The ability to observe quantitatively mechanical events in real time of biological phenomena is an important contribution
of the Optical Tweezers technique for life sciences. The measurements of any mechanical property involves force
measurements, usually performed using a microsphere as the force transducer. This makes the understanding of the
photonic force theory critical. Only very sensitive and precise experimental 3D photonic force measurements for any
particle size will be able to discriminate between different theoretical models. In particular it is important to obtain the
whole photonic force curve as a function of the beam position instead of isolate particular points. We used a dual trap in
an upright standard optical microscope, one to keep the particle at the equilibrium position and the other to disturb it.
With this system we have been able to obtain these force curves as a function of x, y and z position, incident beam
polarization and wavelength. We investigated the optical forces for wavelengths in and out of Mie resonances of
dielectric microspherical cavities for both TM and TE modes and compared the experimental results with the
calculations performed with different models for the optical force.
Partial wave decomposition of incident beams is the first task to be performed to impose boundary conditions at the particle interface in the calculation of the scattering of spherical particles. The coordinate's origin must be in the center of the particle and not at high symmetry positions of the beam. This can be a quite complicated problem, especially when a full vectorial diffraction description of the electromagnetic fields and highly focused laser beams are required where the paraxial limit fails. Traditional approximation techniques have been used to proceed forward and to obtain numerical results. The main fault relies on a radial dependence of the beam shape coefficients, which limits the validity of such approximations. Here we prove that the radial dependence will emerge from the solid angle integration in this way obtaining an exact, closed expression, without any approximation, for the beam shape coefficients, for an arbitrary beam shape, origin and polarization, the special case of a Gaussian beam is presented.
Fluorescent semiconductor nanocrystals in quantum confinement regime (quantum dots) present several well known features which make them very useful tools for biological labeling purposes. Low photo-bleaching rates, high chemical stability, active surface allowing conjugation to living cells, explains the success of this labeling procedure over the commonly used fluorescent dyes. In this paper we report the results obtained with high fluorescent core-shell CdTe-CdS (diameter = 3-7 nm) colloidal nanocrystals synthesized in aqueous medium and conjugated to glucose molecules, incubated with living yeast cells, in order to investigate their glucose up-take activity.
Since optical tweezers trapped microspheres can be used as an ultrasensitive force measurements technique, the knowledge of its theoretical description is of utmost importance. However, even the description of the incident electromagnetic fields under very tight focusing, typical of the optical trap, is not yet a closed problem. Therefore it is important to experimentally obtain whole accurate curves of the force as a function of wavelength, polarization and incident beam 3D position with respect to the center of the microsphere. Theoretical models for optical forces such as the Generalized Lorenz-Mie theory, can then be applied to the precisely evaluated experimental results. Using a dual trap in an upright standard optical microscope, one to keep the particle at the equilibrium position and the other to disturb it we have been able to obtain these force curves as a function of x, y and z position, incident beam polarization and also wavelength. Further investigation of optical forces was conducted for wavelengths in and out Mie resonances of the dielectric microspherical cavities for both TM and TE modes.
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