Several models have been used to calculate optical forces since the beginning of optical trapping in the 1970´s to the Optical Tweezers (OT) in 1986 and are still under discussion these days. These models range from ray tracing geometrical optics to full electromagnetic Maxwell stress tensor formalisms, for very small particles in the Rayleigh regimen to tens of micron size particles in the Mie regime, where Mie resonances also appear. There is also a large effort to distinguish gradient vs scattering forces, which are clearly distinguished the geometrical optics frame, by reflected vs refracted beams, but not so easily distinguished in the full electromagnetic formalism. Moreover, Mie formalism was developed for an incident plane wave, which is far from the high numerical aperture (NA) beams used in OT. Even though there are models for high NA axial beams but most optical tweezers systems today use several OT points with inclined beams. Furthermore, the development of Bessel beams and other beams, also require a much more general calculation of the optical forces, as well as a theory for the forces in waveguides and photonic bandgap fibers in which is impossible to speak in terms of ray optics. One of the main difficult to calculate the optical forces in all these systems is the vector spherical wave decomposition of the incident beam. We develop a very general theory to perform this expansion for any beam which allowed us to calculate the forces for any beam and any particle size, and show a criteria to distinguish gradient from scattering forces, and the role of the Mie resonances in these cases. Dependence of the forces on the size of the particles is an important result for the development of optical chromatography. Experimental results in suspended microspheres allow us to compare the results with theory.
Fabiane Leonel Utino, Marina Garcia, Paulo Eduardo Neves Ferreira Velho, Andréa Fernandes Eloy da Costa França, Rafael Fantelli Stelini, Vitor Bianchin Pelegati, Carlos Lenz Cesar, Elemir Macedo de Souza, Maria Letícia Cintra, Gislaine Vieira Damiani
Sarcoidosis and tuberculoid leprosy (TL) are prototypes of granulomatous inflammation in dermatology, which embody one of the histopathology limitations in distinguishing some diseases. Recent advances in the use of nonlinear optical microscopy in skin have enabled techniques, such as second-harmonic generation (SHG), to become powerful tools to study the physical and biochemical properties of skin. We use SHG images to analyze the collagen network, to distinguish differences between sarcoidosis and TL granulomas. SHG images obtained from skin biopsies of 33 patients with TL and 24 with sarcoidosis retrospectively were analyzed using first-order statistics (FOS) and second-order statistics, such as gray-level co-occurrence matrix (GLCM). Among the four parameters evaluated (optical density, entropy, contrast, and second angular moment), only contrast demonstrated statistical significance, being higher in sarcoidosis (p = 0.02; 4908.31 versus 2822.17). The results may indicate insufficient differentiating power for most tested FOS and GLCM parameters in classifying sarcoidosis and TL granulomas, when used individually. But in combination with histopathology (H&E and complementary stains, such as silver and fast acid stains), SHG analysis, like contrast, can contribute to distinguishing between these diseases. This study can provide a way to evaluate collagen distribution in granulomatous diseases.
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.
Understanding the cellular signaling and function at the nano-bio interface can pave the way towards developing next-generation smart diagnostic tools. From this perspective, limited reports detail so far the cellular and subcellular forces exerted by bacterial cells during the interaction with abiotic materials. Nanowire arrays with high aspect ratio have been used to detect such small forces.
In this regard, live force measurements were performed ex-vivo during the interaction of Xylella fastidiosa bacterial cells with InP nanowire arrays. The influence of nanowire array topography and surface chemistry on the response and motion of bacterial cells was studied in detail. The nanowire arrays were also functionalized with different cell adhesive promoters, such as amines and XadA1, an afimbrial protein of X.fastidiosa. By employing the well-defined InP nanowire arrays platform, and single cell confocal imaging system, we were able to trace the bacterial growth pattern, and show that their initial attachment locations are strongly influenced by the surface chemistry and nanoscale surface topography. In addition, we measure the cellular forces down to few nanonewton range using these nanowire arrays. In case of nanowire functionalized with XadA1, the force exerted by vertically and horizontally attached single bacteria on the nanowire is in average 14% and 26% higher than for the pristine array, respectively. These results provide an excellent basis for live-cell force measurements as well as unravel the range of forces involved during the early stages of bacterial adhesion and biofilm formation.
Biofilms can be defined as a community of microorganisms attached to a surface, living embedded in a self- produced matrix of hydrated extracellular polymeric substances (EPS) which comprises most of the biofilm mass. We have recently used an extensive pool of microscopy techniques (confocal fluorescence, electron and scanning probe microscopies) at the micro and nanoscales in order to create a detailed temporal observation of Xylella fastidiosa biofilm formation, using both wild type strain and Green Fluorescent Protein (GFP)-modified cells of this citrus phytopathogen. We have identified three different EPS compositions, as well as their spatial and temporal distribution from single cell to mature biofilm formation stages. In the initial adhesion stage, soluble-EPS (S-EPS) accumulates at cell polar regions and forms a surface layer which facilitates irreversible cell attachment and cell cluster formation. These small clusters are subsequently connected by filamentous cells; further S-EPS surface coverage facilitates cell attachment and form filaments, leading to a floating framework of mature biofilms. The important role of EPS in X.fastidiosa biology was further investigated by imunolabelling experiments to detect the distribution of XadA1 adhesin, which is expressed in early stages of biofilm formation and released in outer membrane vesicles. This protein is located mainly in S-EPS covered areas, as well as on the filaments, indicating a molecular pathway to the enhanced cell attachment previously observed. These results suggest that S-EPS may thus represent an important target for disease control, slow plant colonization by the bacteria, keeping the plant more productive in the field.
Rodrigo de Andrade Natal, Vitor Pelegati, Caroline Bondarik, Guilherme Mendonça, Sophie Derchain, Carmen Lima, Carlos Cesar, Luís Sarian, José Vassallo
Introduction: In breast cancer (BC), desmoplastic reaction, assembled primarily by fibroblasts, is associated with unfavorable prognosis, but the reason of this fact remains still unclear. In this context, nonlinear optics microscopy, including Fluorescence Lifetime Imaging Microscopy (FLIM), has provided advancement in cellular metabolism research. In this paper, our purpose is to differentiate BC cells metabolism with or without contact to desmoplastic reaction. Formalin fixed, paraffin embedded samples were used at different points of hematoxylin stained sections. Methodology: Sections from 14 patients with invasive ductal breast carcinoma were analyzed with FLIM methodology to NAD(P)H and FAD fluorescence lifetime on a Confocal Upright LSM780 NLO device (Carl Zeiss AG, Germany). Quantification of the fluorescence lifetime and fluorescence intensity was evaluated by SPC Image software (Becker &Hickl) and ImageJ (NIH), respectively. Optical redox ratio was calculated by dividing the FAD fluorescence intensity by NAD(P)H fluorescence intensity. Data value for FLIM measurements and fluorescence intensities were calculated using Wilcoxon test; p< 0.05 was considered significant. Results: BC cells in contact with desmoplastic reaction presented a significantly lower NAD(P)H and FAD fluorescence lifetime. Furthermore, optical redox ratio was also lower in these tumor cells. Conclusion: Our results suggest that contact of BC cells with desmoplastic reaction increase their metabolic activity, which might explain the adverse prognosis of cases associated with higher peritumoral desmoplastic reaction.
Recently, light microscopy-based techniques have been extended to live mammalian models leading to the development
of a new imaging approach called intravital microscopy (IVM). Although IVM has been introduced at the beginning of
the last century, its major advancements have occurred in the last twenty years with the development of non-linear
microscopy that has enabled performing deep tissue imaging. IVM has been utilized to address many biological
questions in basic research and is now a fundamental tool that provide information on tissues such as morphology,
cellular architecture, and metabolic status. IVM has become an indispensable tool in numerous areas. This study presents
and describes the practical aspects of IVM necessary to visualize epithelial cells of live mouse mammary gland with
multiphoton techniques.
Colon cancer is one of the most diffused cancers in the Western World, ranking third worldwide in frequency of incidence after lung and breast cancers. Even if it is curable when detected and treated early, a more accurate premature diagnosis would be a suitable aim for both cancer prognostic and treatment. Combined multimodal nonlinear optical (NLO) microscopies, such as two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third harmonic generation (THG), and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation in colon cancer disease.
NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns between normal and malignant human colonic mucosa. Using a set of scoring methods significant differences both in the content, distribution and organization of stroma collagen fibrils, and lifetime components of NADH and FAD cofactors of human colon mucosa biopsies were found. Our results provide a framework for using NLO techniques as a clinical diagnostic tool for human colon cancer, and also suggest that the SHG and FLIM metrics could be applied to other intestinal disorders, which are characterized by abnormal cell proliferation and collagen assembly.
We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.
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.
Collagen fibers are an essential component of the dynamic process of scarring, which accompanies various
diseases. Scar tissue may reveal different morphologic expressions, such as hypertrophic scars or keloids.
Collagen fibers can be visualized by fluorescent light when stained with eosin.
Second Harmonic Generation (SHG) creates a non linear signal that occurs only in molecules without
inversion symmetry and is particularly strong in the collagen fibers arranged in triple helices. The aim of
this study was to describe the methodology for the analysis of the density and texture of collagen in keloids,
hypertrophic scars and conventional scars. Samples were examined in the National Institute of Science and
Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas. The
images were acquired in a multiphoton microscopy LSM 780-NLO Zeiss 40X. Both signals, two-photon
fluorescence (TPEF) and SHG, were excited by a Mai-Tai Ti:Sapphire laser at 940 nm. We used a
LP490/SP485 NDD filter for SHG, and a BP565-610 NDD filter for fluorescence In each case, ten images
were acquired serially (512×512 μm) in Z-stack and joined together to one patchwork-image . Image
analysis was performed by a gliding-box-system with in-house made software. Keloids, hypertrophic scars
and normal scar tissue show different collagen architecture. Inside an individual case differences of the scar
process may be found between central and peripheral parts. In summary, the use of nonlinear optics is a
helpful tool for the study of scars tissue.
M. Andreoli-Risso, A. S. Duarte, T. Ribeiro, P. Bordeaux-Rego, A. Luzo, M. Baratti, J. Adur, A. de Thomaz, V. Pelegati, H. Carvalho, C. Cesar, P. Kharmadayan, F. Costa, S. Olalla-Saad
Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a
promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in
cell-based therapies. However, conventional slow freezing has always been associated with damage and
mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to
observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the
collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation
(SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue
which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic
lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and
characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific
markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation
was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved
collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved
MSCs presented a directional trend in the collagen fibers formed which was absent in the
cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier
Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant
difference.
P. Bordeaux-Rego, M. Baratti, A. S. Duarte, T. Ribeiro, M. Andreoli-Risso, B. Vidal, J. Miranda, J. Adur, A. de Thomaz, V. Pelegati, F. Costa, H. Carvalho, C. Cesar, A. Luzo, S. Olalla Saad
Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC)
transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the
aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG)
microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The
experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the
microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks,
the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the
lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the
cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers
are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a
greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study
demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based
cell therapy for cartilage repair.
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.
The use of photonics has improved our understanding of biologic phenomena. For the study of the normal and
pathologic architecture of the aorta the use of Two-Photon Excited Fluorescence (TPEF) and Second
Harmonic Generation showed interesting details of morphologic changes of the elastin-collagen architecture
during aging or development of hypertension in previous studies. In this investigation we tried to apply
fluorescence lifetime imaging (FLIM) for the morphologic analysis of human aortas. The aim of our study
was to use FLIM in non-stained formalin-fixed and paraffin-embedded samples of the aorta ascendants in
hypertensive and normotensive patients of various ages, examining two different topographical regions. The
FLIM-spectra of collagen and elastic fibers were clearly distinguishable, thus permitting an exact analysis of
unstained material on the microscopic level. Moreover the FLIM spectrum of elastic fibers revealed variations
between individual cases, which indicate modifications on a molecular level and might be related to FLIM age
or diseases states and reflect modifications on a molecular level.
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.
Nonlinear microscopy imaging technique enable take both images of collagen fibers in dermis through second harmonic
generation (SHG) signal and elastic fibers by two-photon emission fluorescence microscopy (TPEFM). These techniques
are the most commonly used technique for turbid and thick tissue imaging and also to image biological samples which
presents highly ordered structural proteins without any exogenous label. The objective of this study is characterizing
dermis of third-degree burned skin by TPEFM and SHG technique. The modelocked laser (Spectra Physics) source used
in this study with pulse width of approximately 100 fs at 80 MHz was directed into a multiphoton microscope using a
laser scanning unit (Olympus Fluoview 300), mounted on an inverted confocal system microscope (Olympus IX81), with
focusing objective (40x, NA = 1.30). The samples were obtained from Wistar rats, male, adult. One dorsum area was
submitted to burn caused by vapour exposure. The biopsies obtained were cryosectioned in slices of 20 μm width.
Selected area of interface between the injured and healthy subdermal burned skin were imaged by TPEFM and SHG
technique. Two different autofluorescence signals are observed as a function of excitation wavelength. The
autofluorescence observed at 760 nm and 690 nm suggest components of extracellular matrix at differents depths. In
SHG images, collagen fibers are visible. According to the images obtained, these methodologies can be used to
characterize dermis of burned tissue as its healing process with reduced out-of-plane photobleaching and phototoxicity.
Elastic and collagen fibers are essential components of the aorta, the remodeling of
these structures is accompanied with aging in various diseases and life-threatening events. While the
elastic fibers confer resilience to major blood vessels collagen confers resistance to the same.
Elastic fibers are easily visualized in the fluorescent light when stained with hematoxylin
eosin. Second Harmonic Generation (SHG) is a non linear signal that occurs only in molecules
without inversion symmetry and is particularly strong in the collagen fibers arranged in triple helices.
The aim of this paper is to describe the distribution of collagen in the thickness of the thoracic aorta,
and to demonstrate the distribution of between elastic fibers.
The images were acquired in a multifoton microscopy and both signals, Two-phtoton
excitaded fluorescence (TPEF) and SHG, were excited by a Ti:Sapphire laser. We used a band pass
filter to filter the SHG signal from the TPEF signal. The thickness of the aorta varies 2-3 mm, and the
image was composed of the juxtaposition of images of 220 x 220 microns.
We acquired images of a histological slide of the thoracic aorta stained with picrosirius red
(specific for collagen) at a wavelength of 670nm SHG subsequently acquired images with the same
region and observed that the images are overlapping. Therefore, the following images were acquired
by confocal microscopy (fluorescence of eosin for visualization of elastic fibers) and for collagen
SHG. After reconstruction of the images, we observed the distribution of collagen along the aorta.
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.
Metastasis is the main cause of death in cancer patients; it requires a complex process of tumor cell dissemination, extra
cellular matrix (ECM) remodeling, cell invasion and tumor-host interactions. Collagen is the major component of ECM;
its fiber polymerization or degradation evolves in parallel with the evolution of the cancerous lesions. This study aimed
to identify the collagen content, spatial distribution and fiber organization in biopsies of benign and malignant human
ovarian tissues. Biopsies were prepared in slides without dyes and were exposed to 800nm Ti:Sapphire laser (Spectra
Physics, 100 fs pulse duration, 800mW average power, 80MHz repetition rate). The obtained images were recorded at
triplets, corresponding to clear field, multiphoton and second harmonic generation (SHG) mycroscopy. Data showed
considerable anisotropy in malignant tissues, with regions of dense collagen arranged as individual fibers or in
combination with immature segmental filaments. Radial fiber alignment or regions with minimal signal were observed in
the high clinical grade tumors, suggesting degradation of original fibers or altered polymerization state of them. These
findings allow us to assume that the collagen signature will be a reliable and a promising marker for diagnosis and
prognosis in human ovarian cancers.
Elastic fibers are essential components of the human aorta, and there is an association between
elastin fibers remodeling and several diseases. Hypertension is one such example of a disease leading to
elastin fibers remodeling. These fibers can be easily seen in
eosin-hematoxilin (HE) stained histologic
sections when observed by UV-excited fluorescence microscopy or by a much more precise Laser Scanning
Confocal Microscope (LSCM). In order to study the effect of the hypertension on the elastin fibers pattern we
developed an automatic system (software and hardware) to count the number of elastin fibers and to measure
the distance between them in a LSCM and used it compare the statistical distribution of the distance between
these fibers in normotensive and hypertensive patients. The full image of the whole sample (2 or 3mm long)
was composed by several 220×220μm frames with 512×512 pixels. The software counters fiber and distance
between fibers. We compared the elastic fiber texture in routinely
HE-stained histologic slides of the aorta
ascendens in 24 normotensive and 30 hypertensive adult patients of both sexes and of similar age from our
autopsy files. Our results show that the average number of fibers is the same for both cases but the distance
between the fibers are larger for hypertensive patients than for normotensive ones.
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.
We report the fabrication of glass multilayer doped with semiconductor nanoparticles. The glass
matrix was fabricated by Plasma Enhanced Chemical Deposition (PECVD using tetramethoxysilane
(TMOS) as precursor. The RF power was supplied by a RF-150 TOKYO HI-Power operating at
13.56 MHz and coupled to the RF electrodes through a matching box. The nanoparticles were
grown by pulsed laser deposition (PLD) of a PbTe target using the second harmonic of a Q-Switched
Quantel Nd:YAG laser in high purity inert gas atmosphere.
The influence of gas and background pressure and in the nanoparticle size and size distribution is
studied. The morphological properties of the nanostructured material were studied by means of High
Resolution Transmission Electron Microscopy(HRTEM), grazing-incidence smallangleX-ray
scattering (GISAXS).
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.
Elastic fibers are an important component of many organs and tissues, such as
skin, lungs, arteries, ligaments, intervertebral discs and cartilage Their function is to
endow tissues with elastic recoil and resilience, to act as an important adhesion
template for cells, and to regulate growth factor availability (1,2). Loss or remodeling of
the elastic fiber texture occurs in many diseases. Degeneration and fragmentation of
elastic fibers and aging are intimately related (3). Recently, the importance of elastin for
the study of malignant tumor progression has been emphasized (4,5). Elastic tissue
may be a significant reservoir of angiostatic molecules and soluble elastin as well as
elastin peptides, that are inhibitors of the metastatic process in experimental tumor
models (4). Elastic fibers are involved in the anatomic remodeling of chronic pulmonary
diseases (6) and, especially, of diseases of the arterial wall (7, 8). The study of these
phenomena is important for the understanding of the pathophysiologic basis of the
diseases. Recently the role of elastic fibers in small diameter vascular graft design has
been emphasized (2). The possibility to regenerate or engineer elastic fibres and
tissues creates an important challenge, not only to understand the molecular basis of
elastic-fibre biology (1,2), but also of its spatial arrangement and remodeling in the
diseased tissues. Subtle changes of the complex elastic fiber network may be involved
in the pathogenesis of diseases. Therefore a precise and objective histopathologic
description is necessary.
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.
Laser ablation (LA) is a thin film fabrication technique which has generated a lot
of interest in the past few years as one of the simplest and most versatile methods for the
deposition of a wide variety of materials. With the rapid development experienced in the
generation of ultra short laser pulses, new possibilities were opened for the laser ablation
technique, using femtosecond lasers as ablation source. It is commonly believed that
when the temporal length of the laser pulse became shorter than the several picoseconds
required to couple the electronic energy to the lattice of the material, thermal effects
could not play a significant role. Since the pulse width is too short for thermal effects to
take place, with each laser pulse a few atom layers of material are direct vaporized away
from the target surface and a better control in the quantum dots (QDs) fabrication could
be achieved.
In this work we report the fabrication of PbTe QDs by femtosecond laser ablation of a
PbTe target in argon atmosphere. Experiments were carried out using a typical LA
configuration comprising a deposition chamber and an ultra short pulsed laser (100 fs; 30
mJ) at a central wavelength of 800 nm. PbTe was chosen because its QDs absorption
band can be controlled by its size to fall in the spectral window of interest for optical
communications (1.3-1.5 μm). This, together with the QD high optical nonlinearity,
makes this material an excellent candidate for development of photonic devices.
It was investigated the influence of the number of laser pulses in the formation of the
nanoparticles. The structural parameters and the surface density of the nanoparticles were
studied by high resolution transmission electron microscopy (HRTEM).
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.
In the past it was observed that buck ball doped glasses showed enhanced optical
nonlinearities. However, carbon nanotubes are much more stable than buck ball and
should be a better choice for that purpose. Therefore we decided to investigate the
possibility to produce carbon nanotubes doped tellurite glasses and measured their
optical nonlinearities. Tellurite glasses already have a larger nonlinearity compared to
silica, and other, glasses. We produced TeO2-ZnO tellurite family glasses doped with
multi wall Carbon Nanotube (CNT). The CNTs acquired from Carbolex were
vigorously mechanically mixed with the tellurite glass precursors and melted in
platinum crucible around 650°C in a controlled atmosphere inside an electrical induction
furnace. We used the lowest temperature possible and controlled atmosphere to avoid
the CNT oxidation. The glass melt was cast in a stainless steel and thermally treated at
300°C for 5 hours to relieve internal stresses. The samples were than cutted and
polished to perform the optical characterization. We measured refractive index and
thermo physical properties, such as vitreous transition Tg, crystallization onset Tx and
melting Tf temperatures. Raman spectroscopy showed the possible presence of CNTs.
Red blood cell (RBC) aggregation in the blood stream is prevented by the zeta potential created by its negatively charged membrane. There are techniques, however, to decrease the zeta potential and allow cell agglutination, which are the basis of most of antigen-antibody tests used in immunohematology. We propose the use of optical tweezers to measure membrane viscosity, adhesion, zeta potential, and the double layer thickness of charges (DLT) formed around the cell in an electrolytic solution. For the membrane viscosity experiment, we trap a bead attached to RBCs and measure the force to slide one RBC over the other as a function of the velocity. Adhesion is quantified by displacing two RBCs apart until disagglutination. The DLT is measured using the force on the bead attached to a single RBC in response to an applied voltage. The zeta potential is obtained by measuring the terminal velocity after releasing the RBC from the trap at the last applied voltage. We believe that the methodology proposed here can provide information about agglutination, help to improve the tests usually performed in transfusion services, and be applied for zeta potential measurements in other samples.
In this work we report the fabrication of PbTe quantum dots multilayers embedded in SiO2 by
alternatively use of Laser Ablation and Plasma Enhanced Chemical Vapor Deposition
techniques. The quantum dots were grown by pulsed laser deposition (PLD) of a PbTe target
using the second harmonic of a Q-Switched Quantel Nd:YAG laser in high purity argon
atmosphere. The glass matrix was fabricated by PECVD using tetramethoxysilane (TMOS) as
precursor. The RF power was supplied by a RF-150 TOKYO HI-Power operating at 13.56 MHz
and coupled to the RF electrodes through a matching box. The deposition rates as well as the best
growth parameters for both the nanoparticles and the glass matrix were obtained from a previous
work. The morphological properties of the nanostructured material were studied by means of igh
Resolution Transmission Electron Microscopy(HRTEM), grazing-incidence small-angle X-ray
scattering (GISAXS) and X-ray reflectometry . Unlike HRTEM, which extracts information of a
submicron region of the sample and only a few thousand particles are observed, GISAXS signal
is obtained through an average over orders of magnitude larger number of particles (perhaps 1012
particles) distributed over an area of tens of square millimeters. This fact means that GISAXS
sampling is much more representative of the sample as whole.
Finally, multilayers were grown inside a Fabry-Perot cavity. The complete system operates as
an optical switching device for the infrared region. The device was characterized by Scanning
Electron Microscopy and optical absorption.
PbTe thin films were grown on BK7 glass and Si(100) substrates using femtosecond pulsed laser deposition at room temperature. The influence of the background pressure and the laser fluence on the structural and optical characteristics of the PbTe films was
studied. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were used to characterize the surface and structural properties of the deposited PbTe thin films, respectively. Transmission spectroscopy measurements in the visible and infrared region
(VIS-IR) were used to investigate the optical properties of the PbTe thin films.
The red blood cell (RBC) viscoelastic membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that are responsible for cell agglutination. Manipulating RBCs rouleaux with a double optical tweezers, we observed that the cells slide easily one over the others but are strongly connected by their edges. An explanation for this behavior could be the fact that when the cells slide one over the others, proteins are dragged through the membrane. It confers to the movement a viscous characteristic that is dependent of the velocity between the RBCs and justifies why is so easy to slide them apart. Therefore, in a first step of this work, by measuring the force as a function of the relative velocity between two cells, we confirmed this assumption and used this viscous characteristic of the RBC rouleaux to determine the apparent membrane viscosity of the cell. As this behavior is related to the proteins interactions, we can use the apparent membrane viscosity to obtain a better understanding about cell agglutination. Methods related to cell agglutination induced by antigen-antibody interactions are the basis of most of tests used in transfusion centers. Then, in a second step of this work, we measured the apparent membrane viscosity using antibodies. We observed that this methodology is sensitive to different kinds of bindings between RBCs. Better comprehension of the forces and bindings between RBCs could improve the sensibility and specificity of the hemagglutination reactions and also guides the development of new potentiator substances.
The research in biomedical photonics is clearly evolving in the direction of the understanding of biological
processes at the cell level. The spatial resolution to accomplish this task practically requires photonics tools.
However, an integration of different photonic tools and a multimodal and functional approach will be necessary to
access the mechanical and biochemical cell processes. This way we can observe mechanicaly triggered biochemical
events or biochemicaly triggered mechanical events, or even observe simultaneously mechanical and biochemical
events triggered by other means, e.g. electricaly. One great advantage of the photonic tools is its easiness for
integration. Therefore, we developed such integrated tool by incorporating single and double Optical Tweezers with
Confocal Single and Multiphoton Microscopies. This system can perform 2-photon excited fluorescence and Second
Harmonic Generation microscopies together with optical manipulations. It also can acquire Fluorescence and SHG
spectra of specific spots. Force, elasticity and viscosity measurements of stretched membranes can be followed by
real time confocal microscopies. Also opticaly trapped living protozoas, such as leishmania amazonensis.
Integration with CARS microscopy is under way. We will show several examples of the use of such integrated
instrument and its potential to observe mechanical and biochemical processes at cell level.
The red blood cell (RBC) viscoelastic membrane contains proteins and glycolproteins embedded in, or attached, to a
fluid lipid bilayer and are negatively charged, which creates a repulsive electric (zeta) potential between the cells
and prevents their aggregation in the blood stream. The basis of the immunohematologic tests is the interaction
between antigens and antibodies that causes hemagglutination. The identification of antibodies and antigens is of
fundamental importance for the transfusional routine. This agglutination is induced by decreasing the zeta-potential
through the introduction of artificial potential substances. This report proposes the use of the optical tweezers to
measure the membrane viscosity, the cell adhesion, the zeta-potential and the size of the double layer of charges
(CLC) formed around the cell in an electrolytic solution. The adhesion was quantified by slowly displacing two
RBCs apart until the disagglutination. The CLC was measured using the force on the bead attached to a single RBC
in response to an applied voltage. The zeta-potential was obtained by measuring the terminal velocity after releasing
the RBC from the optical trap at the last applied voltage. For the membrane viscosity experiment, we trapped a bead
attached to RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. After
we tested the methodology, we performed measurements using antibody and potential substances. We observed that
this experiment can provide information about cell agglutination that helps to improve the tests usually performed in
blood banks. We also believe that this methodology can be applied for measurements of zeta-potentials in other kind of samples.
A new polymeric open optical fiber with a star cross section from extruded PMMA grains was
developed. We have painted the external surface of this fiber with the Europium chelate or
Antracene. We have observed the typical luminescence of these compounds when excited with
nanosecond 355 nm pulsed laser. These results show that the developed fiber presents potentialities
for optical sensing.
Tellurite glasses are important as a host of Er3+ ions because of their great solubility and because they present broader
gain bandwidths than Er3+-doped silica, with promise to increase the bandwidth of communication systems. However,
the small glass stability range (GSR) of tellurite glasses compromises the quality of the optical fibers. We show that the
addition of CsCl to tellurite glasses can increase their GSR, making it easier to draw good quality optical fibers. CsCl
acts as a network modifier in glass systems, weakening the network by forming Te-Cl bonds. We show that the thermal
expansion coefficient mismatch is in the right direction for optical fiber fabrication purposes and that the Bi2O3 content can be used to control the refractive index of clad and core glasses. Single-mode and multi-mode Er3+-doped optical
fibers were produced by the rod-in-tube method using highly homogeneous TeO2-ZnO-Li2O-Bi2O3-CsCl glasses. Far infrared spectra of the glass samples exhibit absorption bands of the Te-Cl bond.
We have been able to produce soft glass conventional core-clad and micro-structured fibers using rod-and-tube and stack-and-draw method respectively. The stack-and-draw technique shows several difficulties when used with soft glasses, that we managed to avoid using two different lead and alkaline glasses. Non commercial glasses and fibers were thermo-mechanically and optically characterized.
Chemotaxis is the mechanism microorganisms use to sense the environment surrounding them and to direct their
movement towards attractive, or away from the repellent, chemicals. The biochemical sensing is almost the only way for
communication between unicellular organisms. Prokaryote and Eukaryote chemotaxis has been mechanically studied
mainly by observing the directionality and timing of the microorganisms movements subjected to a chemical gradient,
but not through the directionality and strength of the forces it generates. To observe the vector force of microorganisms
under a chemical gradient we developed a system composed of two large chambers connected by a tiny duct capable to
keep the chemical gradient constant for more than ten hours. We also used the displacements of a microsphere trapped in
an Optical Tweezers as the force transducer to measure the direction and the strength of the propulsion forces of
flagellum of the microorganism under several gradient conditions. A 9μm diameter microsphere particle was trapped
with a Nd:YAG laser and its movement was measured through the light scattered focused on a quadrant detector. We
observed the behavior of the protozoa Leishmania amazonensis (eukaryote) under several glucose gradients. This
protozoa senses the gradient around it by swimming in circles for three to five times following by tumbling, and not by
the typical straight swimming/tumbling of bacteria. Our results also suggest that force direction and strength are also
used to control its movement, not only the timing of swimming/tumbling, because we observed a higher force strength
clearly directed towards the glucose gradient.
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.
Multilayers of PbTe quantum dots embedded in SiO2 were fabricated by alternatively use of Laser
Ablation and Plasma Enhanced Chemical Vapor Deposition techniques. A set o samples containing
different PbTe nanoparticles sizes was prepared for the study. The morphological properties of the
nanostructured material were studied by means of grazing-incidence small-angle X-ray scattering
(GISAXS) and x-ray reflectometry (XRR) techniques. A preliminary analysis of the GISAXS
spectra provided information about the multilayer periodicity and its relationship to the size of the
deposited PbTe nanoparticles.
Multilayers of PbTe quantum dots embedded in SiO2 were fabricated and characterized by
means of Fourirer transform infrared and x-ray spectrometry and transmission electron
microscopy. The quantum dots were grown by laser ablation of a PbTe target using the second
harmonic of a Q-Switched Quantel Nd:YAG laser under high purity argon atmosphere. The
glass matrix was fabricated by Plasma Enhanced Chemical Vapor Deposition using
tetramethoxysilane as precursor. The reason for choosing PbTe was the absorption bands this
material exhibits in the region of interest for optical communications 1.3-1.5μm making this
material an excellent candidate for development of optical devices.
For the glass matrix, it was studied the influence of growing parameters like RF power,
distance between the RF electrodes and the total pressure in the properties of the SiO2 films. The
parameters for the PbTe ablation were assumed from a previous work. FTIR and refractive index
measurements were used to estimate the best growth parameters for the dielectric host.
TMOS partial pressure proved to be an important parameter to diminish the nanoparticle
coalescence during the multilayer fabrication. Multilayer X-ray diffraction patterns were used to
estimate the nanoparticles diameter. Morphological properties of the nanostructured material
were studied using transmission electron microscopy.
The displacements of a dielectric microspheres trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences. This system can measure forces on the 50 femto Newtons to 200 pico Newtons range, of the same order of magnitude of a typical forces induced by flagellar motion. The process in which living microorganisms search for food and run away from poison chemicals is known is chemotaxy. Optical tweezers can be used to obtain a better understanding of chemotaxy by observing the force response of the microorganism when placed in a gradient of attractors and or repelling chemicals. This report shows such observations for the protozoa Leishmania amazomenzis, responsible for the leishmaniasis, a serious tropical disease. We used a quadrant detector to monitor the movement of the protozoa for different chemicals gradient. This way we have been able to observe both the force strength and its directionality. The characterization of the chemotaxis of these parasites can help to understand the infection mechanics and improve the diagnosis and the treatments employed for this disease.
We present degenerate and nondegenerate two-photon absorption spectra in a series of CdSe and CdTe quantum dots. The measurements show that the two-photon absorption (2PA) spectrum is strongly dependent on the quantum dot size and that the 2PA coefficient decreases as the quantum dot size decreases, and it is larger for the frequency nondegenerate process. Previously we had shown a theoretical analysis of these results based on a simple model using the effective mass approximation. Although this model works well for larger quantum dots, it fails for the smaller ones. Here we use the more (formula available in manuscript) model for the band structure and consider the hole band mixing in quantum dots to describe our data. This theory better describes the spectral structures for smaller quantum dots and also predicts the decrease of the 2PA coefficient with the decrease of quantum dot size. This is due to the reduction of the number of possible transitions and the blue shift of the optical bandgap from quantum confinement. This theory predicts the reduction of the 2PA coefficient with size, although our experimental results show an even stronger reduction.
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.
The fluid lipid bilayer viscoelastic membrane of red blood cells (RBC) contains antigen glycolproteins and proteins which can interact with antibodies to cause cell agglutination. This is the basis of most of the immunohematologic tests in blood banks and the identification of the antibodies against the erythrocyte antigens is of fundamental importance for transfusional routines. The negative charges of the RBCs creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. The first counterions cloud strongly binded moving together with the RBC is called the compact layer. This report proposes the use of a double optical tweezers for a new procedure for measuring: (1) the apparent membrane viscosity, (2) the cell adhesion, (3) the zeta potential and (4) the compact layer's size of the charges formed around the cell in the electrolytic solution. To measure the membrane viscosity we trapped silica beads strongly attached to agglutinated RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. The RBC adhesion was measured by slowly displacing two RBCs apart until the disagglutination happens. The compact layer's size was measured using the force on the silica bead attached to a single RBC in response to an applied voltage and the zeta potential was obtained by measuring the terminal velocity after releasing the RBC from the optical trap at the last applied voltage. We believe that the methodology here proposed can improve the methods of diagnosis in blood banks.
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.
Micro-structured Er3+ - Tm3+ co-doped tellurite fiber with three rings of holes was
fabricated using a soft glass drawing tower by a stack-and-draw technique. Amplified
spontaneous emission (ASE) around 1550nm band were observed when pumped with both,
980nm and 790nm, lasers.
In this work we present the preparation, characterization and conjugation of colloidal core shell CdS-Cd(OH)2 quantum dots to health and cancer glial rats living cells in culture media. The particles were obtained via colloidal synthesis in aqueous medium, with final pH=7.3-7.4. Laser Scan Confocal Microscopy (LSCM) and Fluorescence Microscopy were used to evaluate fluorescence intensities and patterns of health and cancer (glioblastoma) glial cells labeled with the quantum dots in different time intervals. Health and cancer glial cells clearly differ in their fluorescence intensities and patterns. These different fluorescence intensities and patterns may be associated to differences concerning cellular membrane and metabolic features of health and cancer cells. The results obtained indicate the potential of the methodology for fast and precise cancer diagnostics.
This work reports the fabrication of planar and channel waveguides by Ag+ → Na+ ion exchange in an Er3+ doped tellurite glass with a composition of 75TeO2-2GeO2-10Na2O-12ZnO-1Er2O3 (mol %). The glass was chemically stable during the ion-exchange process. We have been able to produce single and multi-mode planar waveguides controlling the depths of the waveguides by varying ion-exchange temperatures, from 250 to 280 °C, and times, from 3 to 12 h. We also show preliminary results of channel waveguide fabrication with the same technique. The waveguide effective refractive index curves and attenuation (11 dB/cm) at 1536 nm were measured with a Metricom prism coupler. The Amplified Spontaneous Emission (ASE) spectra showed a 152 nm bandwidth when pumped with 120 mW laser pump at 980 nm.
Thermal Lens (TL) and spectroscopic characterizations were performed in 70TeO2-19WO3-7Na2O-4Nb2O5 (mol%) tellurite glasses. TL measurements were accomplished in Er3+ /Tm3+ co-doped tellurite glasses in function of the Tm2O3 concentration (0.4-1.6 x1020 ions/cm3). Fluorescence spectra at 488 nm showed that Er3+ /Tm3+ co-doped tellurite glasses present several emission bands between (500-1800) nm. However, the more intense emission bands correspond to the Tm3+ and Tm3+ transitions (4I13/2 → 4I15/2 and 3F4 → 3H6), respectively. The absolute nonradiative quantum efficiency (φ) was determined by TL method. Higher values of φ were obtained with the increase of Tm2O3 concentration inside of the Er3+/Tm3+ co-doped tellurite glasses. These results are corroborated by the Judd-Ofelt calculations.
In this work we used a Thermal Mechanic Analysis equipment to produce the channel FOG waveguides by pressing an Er3+ doped tellurite glass optical fiber against one Er3+ ion doped tellurite glass substrate kept under Tc ± 30 oC (Tc = soft point). The luminescence and waveguide refractive index were measured. Scanning electron microscopy was used to observe the obtained structure. The objective is to produce a new concept in components of integrated optical circuits. Then this work report the production of Er3+-doped tellurite glass channel waveguides using the novel concept of Benson et al[1] of fiber on glass (FOG). To succeed with this technique it is important to correlate the main thermo-physical characteristics of the substrate and the fiber, which are the transition temperature Tg, the temperature of the onset of crystallization Tx, the maximum crystallization temperature Tc and the thermal expansion coefficient. The Tg, Tx and Tc values were determined by Differential Thermal Analysis (DTA), while the thermal expansion coefficient was determined by Thermal Mechanical Analysis (TMA). For the FOG purpose the thermal stability range, Tx - Tg, is an important temperature region which defines if the glass will have enough viscosity to shape in the FOG concept.
Er3+-Tm3+ co-doped tellurite photonic crystal fiber was fabricated via a stack-and-draw procedure and without using extrusion in any stage. The final fiber presents a 187 nm bandwidth of amplified spontaneous emission (ASE) intensity around 1550nm when pumped with 790nm. In this manuscript a soft-glass tube fabrication technique, using the centrifugation method, is also shown.
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.
The red blood cell (RBC) viscoelastic membrane contains proteins and glycolproteins embedded in, or attached, to a fluid lipid bilayer and are negatively charged, which creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. There are techniques, however, to decrease the zeta potential to allow cell agglutination which are the basis of most of the tests of antigen-antibody interactions in blood banks. This report shows the use of a double optical tweezers to measure RBC membrane viscosity, agglutination and zeta potential. In our technique one of the optical tweezers trap a silica bead that binds strongly to a RBC at the end of a RBCs rouleaux and, at the same time, acts as a pico-Newton force transducer, after calibration through its displacement from the equilibrium position. The other optical tweezers trap the RBC at the other end. To measure the membrane viscosity the optical force is measured as a function of the velocity between the RBCs. To measure the adhesion the tweezers are slowly displaced apart until the RBCs disagglutination happens. The RBC zeta potential is measured in two complimentary ways, by the force on the silica bead attached to a single RBC in response to an applied electric field, and the conventional way, by the measurement of terminal velocity of the RBC after released from the optical trap. These two measurements provide information about the RBC charges and, also, electrolytic solution properties. We believe this can improve the methods of diagnosis in blood banks.
In this work we applied core-shell CdS/Cd(OH)2 quantum dots (QDs) as fluorescent labels in the Leishmania amazonensis protozoarium. The nanocrystals (8-9 nm) are obtained via colloidal synthesis in aqueous medium, with final pH=7 using sodium polyphosphate as the stabilizing agent. The surface of the particles is passivated with a cadmium hydroxide shell and the particle surface is functionalized with glutaraldehyde. The functionalized and non-functionalized particles were conjugated to Leishmania organisms in the promastigote form. The marked live organisms were visualized using confocal microscopy. The systems exhibit a differentiation of the emission color for the functionalized and non-functionalized particles suggesting different chemical interactions with the promastigote moieties. Two photon emision spectra (λexc=795nm) were obtained for the promastigotes labeled with the functionalized QDs showing a significant spectral change compared to the original QDs suspension. These spectral changes are discussed in terms of the possible energy deactivation processes.
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.
Multilayer of PbTe quantum dots embedded in SiO2 were fabricated by alternatively use of Plasma Enhanced Chemical Vapor Deposition and Laser Ablation techniques. The optimal growing parameters for both the SiO2 films and the PbTe quantum dots were obtained. The refractive index and optical absorption of the sample were studied. Multilayer X-ray diffraction patterns were used to estimate the nanoparticles diameter. Morphological properties of the nanostructured material were studied using Transmission Electron Microscopy. Both absorption spectra and X-ray diffraction patterns reveled the nanoparticles are 6-8 nm in diameter, consequently appropriate for developing optical devices in the infra red region. Finally the multilayer was grown inside a Fabry Perot cavity. The transmittance of the one-dimensional photonic crystal was measured.
In this work we developed a setup consisting of an Optical Tweezers equipped with linear and non-linear micro-spectroscopy system to add the capabilities of manipulation and analysing captured objects. Our setup includes a homemade confocal spectrometer using a monochromator equipped with a liquid nitrogen cooled CCD. The spectroscopic laser system included a cw and a femtosecond Ti:sapphire lasers that allowed us to perform Raman, hyper-Raman, hyper-Rayleigh and two photon Excited (TPE) luminescence in particles trapped with an Nd:YAG cw laser. We obtained Raman spectra of a single trapped polystyrene microsphere and a single trapped red blood cell to evaluate the performance of our system. We also observed hyper-Rayleigh and hyper-Raman peaks for SrTiO3 with 60s integration time only. This was possible because the repetition rate of the femtosecond Ti:sapphire lasers, on the order of 80 MHz, are much higher than the few kHz typical picosecond laser repetition rate used before in hyper- Raman experiment, which required acquisition times of order of few hours. We used this system to perform scanning microscopy and to acquire TPE luminescence spectra of captured single stained microsphere and cells conjugated with quantum dots of CdS and CdTe and hyper-Rayleigh spectra of a noncaptured ZnSe microparticle. The results obtained show the potential presented by this system and fluorescent labels to perform spectroscopy in a living trapped microorganism in any neighbourhood and dynamically observe the chemical reactions changes in real time.
The displacements of a polystyrene microsphere trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences such as the measurement of forces of living microorganisms or the viscosity of local fluids. The technique we used allowed us to measure forces on the 200 femto Newtons to 4 pico Newtons range of the protozoa Leishmania amazonensis, responsible for a serious tropical disease. These observations can be used to understand the infection mechanism and chemotaxis of these parasites. The same technique was used to measure viscosities of few microliters sample with agreement with known samples better than 5%. To calibrate the force as a function of the microsphere displacement we first dragged the microsphere in a fluid at known velocity for a broad range of different optical and hydrodynamical parameters. The hydrodynamical model took into account the presence of two walls and the force depends on drag velocity, fluid viscosity and walls proximities, while the optical model in the geometric optics regime depends on the particle and fluid refractive indexes and laser power. To measure the high numerical (NA) aperture laser beam power after the objective we used an integration sphere to avoid the systematic errors of usual power meters for high NA beams. After this careful laser power measurement we obtained an almost 45 degrees straight line for the plot of the optical force (calculated by the particle horizontal displacement) versus hydrodynamic force (calculated by the drag velocity) under variation of all the parameters described below. This means that hydrodynamic models can be used to calibrate optical forces, as we have done for the parasite force measurement, or vice-versa, as we did for the viscosity measurements.
Up to now optical spectroscopies have analyzed the scattered light or the heat generated by absorption as a function of the wavelength to get information about the samples. Among the light matter interaction phenomena one that has almost never been used for spectroscopy is the direct photon momenta transfer. Probably because the forces involved are very small, varying from hundreds of femto to tens of pico Newtons. However, the nowadays very popular Optical Tweezers can easily accomplish the task to measure the photon momenta transfer and may be the basis for the Optical Force Spectroscopy. We demonstrate its potential as such a tool by observing more than eight Mie resonance peaks of a single polystyrene microsphere, and showed the capability to selective couple the light to either the TE, TM or both microsphere modes depending of the beam size, the light polarization and the beam positioning. The Mie resonances can change the optical force values by 30-50%. Our results also clearly show how the beam polarization breaks the usually assumed azimuthal symmetry by Optical Tweezers theories. We also obtained the spectrum from the two photon excited luminescence using the Optical Tweezers to hold a single bead suspended and a femtosecond Ti:sapphire laser for the non-linear excitation. This spectrum shows the pair of peaks due to both TE and TM spherical cavity modes. We have been able to observe more than 14 Mie resonance peaks in the TPE luminescence. Our results are in good agreement with optical force calculations using Maxwell stress tensor and partial wave decomposition of the incident beam approximated to a 3th order gaussian beam.
Both degenerate and nondegenerate two-photon absorption (2PA) spectra are studied in two different samples of CdTe quantum-dots in borosilicate glass hosts. One sample (CdTe-600) contains quantum-dots of radius 3.2 ± 0.2 nm and has its absorption edge at 600nm. The other sample (CdTe-750) contains quantum-dots of radius 6.6 ± 0.9 nm and absorption edge at 750nm. CdTe-600 contains quantum-dots with a narrower size distribution than CdTe-750. Consequently, the peaks corresponding to discrete transitions are more clearly visible in CdTe-600 than in CdTe-750. Both nondegenerate and degenerate spectra for these samples show a marked difference from bulk CdTe. In CdTe-750 the two-photon absorption spectrum has a shape similar to that for bulk solids but for CdTe-600 the 2PA spectrum is somewhat different from that expected for the bulk. In the Z-scan measurements we also observed a photo-darkening effect, which is accompanied by an increase in the measured effective 2PA coefficient. All results suggest that 2PA cannot be predicted by the bulk theory especially near to the 2PA edge, that the 2PA in quantum dots is generally smaller than would be expected for the same volume of bulk semiconductor with the same band edge, and that the quantum-dot size and size distribution play important roles in the 2PA spectral behavior and magnitude.
We have developed a method for measuring the red blood cell (RBC) membrane overall elasticity μ by measuring the deformation of the cells when dragged at a constant velocity through a plasma fluid by an optical tweezers. The deformability of erythrocytes is a critical determinant of blood flow in the microcirculation. We tested our method and hydrodynamic models, which included the presence of two walls, by measuring the RBC deformation as a function of drag velocity and of the distance to the walls. The capability and sensitivity of this method can be evaluated by its application to a variety of studies, such as, the measurement of RBC elasticity of sickle cell anemia patients comparing homozygous (HbSS), including patients taking hydroxyrea (HU) and heterozygous (HbAS) with normal donors and the RBC elasticity measurement of gamma irradiated stored blood for transfusion to immunosupressed patients as a function of time and dose. These studies show that the technique has the sensitivity to discriminate heterozygous and homozygous sickle cell anemia patients from normal donors and even follow the course of HU treatment of Homozygous patients. The gamma irradiation studies show that there is no significant change in RBC elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not, but there was a huge change in the measured elasticity for the RBC units stored for more than 21 days after irradiation. These finds are important for the assessment of stored irradiated RBC viability for transfusion purposes because the present protocol consider 28 storage days after irradiation as the limit for the RBC usage.
We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.
In this work we present the fabrication of tellurite glass photonic crystal fiber doped with a very large erbium concentration. Tellurite glasses are important hosts for rare earth ions due to its very high solubility, which allows up to 10,000 ppm Er3+ concentrations. The photonic crystal optical fibers and tellurite glasses can be, therefore, combined in an efficient way to produce doped fibers for large bandwidth optical amplifiers. The preform was made of a 10 mm external diameter tellurite tube filled with an array of non-periodic tellurite capillaries and an erbium-doped telluride rod that constitute the fiber core. The preform was drawn in a Heathway Drawing Tower, producing fibers with diameters between 120 - 140 μm. We show optical microscope photography of the fiber’s transverse section. The ASE spectra obtained with a spectra analyzer show a red shift as the length of the optical fiber increases.
Thin films of glass doped with PbTe quantum dots were successfully fabricated. The semiconducting quantum dots were grown by laser ablation of a PbTe target (99.99%) using the second harmonic of a Q-Switched Quantel Nd:YAG laser under high purity argon atmosphere. The glass matrix was fabricated by a plasma chemical vapor deposition method using vapor of tetramethoxysilane (TMOS) as precursor. The QD's and the glass matrix were alternately deposited onto a Si (100) wafer for 60 cycles. Cross-section TEM image clearly showed QD's layer well separated from each other with glass matrix layers. The influence of the ablation time on the size distribution of the quantum dots is studied. HRTEM revealed anisotropy in the size of the QD's: they were about 9nm in the high and 3-5 in diameter. Furthermore HRTEM studies revealed that the QD's basically growth in the (200) and (220) directions. The thickness of the glass matrix layer was about 20 nm. Absorption, photo luminescence and relaxation time of the multilayer were also measured.
We present a simple and efficient method for marking living human red blood cells using CdS (Cadmium Sulfide) quantum dots (QDs). The nanocrystals were obtained via colloidal synthesis in aqueous medium with final pH=7 using sodium polyphosphate as the stabilizing agent. The methodology implementation is simple, do not requires additional capping layers nor narrow size QDs distribution. The synthesized nanoparticles were conjugated to monoclonal A anti-body. The resulting conjugates QDs/anti-A were incubated with human erythrocytes of blood groups A and O for 30 min at 37°C. The living cells in contact with the quantum dots maintained their properties for several days showing the low level of citotoxicity of the quantum dots. The conjugation of CdS QDs/anti-A show simultaneous red and green fluorescence when excited with 543 and 488 nm respectively. The efficiency of the conjugation QDs/anti-body to the erythrocytes, for each system, was monitored by confocal microscopy. The comparative analysis of the micrographs was done with the luminescence intensity maps of the samples obtained under constant capture conditions, such as, pinhole, filters, beam splitters and photomultiplier gain. The conjugates QDs/anti-A intensely marked group A erythrocytes and did not show any luminescence for group O erythrocytes, showing the sensitivity of the labeling procedure. In conclusion, we show the viability of the use of high luminescent and stable quantum dots as fluorescent labels for human erythrocytes with a methodology of simple implementation and the possibility to use them to distinguish different blood groups.
Semiconductor nanocrystals composed by few hundred to a few thousand atoms also known as quantum dots have received substantial attention due to their size tunable narrow-emission spectra and several other advantages over organic molecules as fluorescent labels for biological applications, including resistence to photodegradation, improved brightness and only one laser excitation that enable the monitoring of several processes simultaneously. In this work we have synthesized and characterized thiol-capped CdTe and bioconjugated them to macrophages. We have mapped the fluoroscence intensity along the macrophage's body in our set up consisting of an optical tweezer plus a non-linear micro-spectroscopy system to perform scanning microscopy and observe spectra using two photon excited luminescence.
Tellurite glasses optical fibers became promising for optical amplifiers due to its high rare earth ions solubility and very large amplification bandwidth. Among several tellurite glasses the TeO2-WO3-Na2O-Nb2O5 system show one of the largest bandwidth. Our previous characterization of lifetime using the omega2, omega4, omega6, Judd-Ofelt parameters indicate a quantum efficiency maximum for 7500ppm Er3+ concentration. Therefore we decided to produce jointed Er3+ and Tm3+ single mode optical fibers with this glass system keeping the 7500ppm Er3+ concentration and varying the Tm3+ concentration from 2500ppm to 15000ppm. This single mode fiber was pumped by 120mW of the semiconductor laser at 790nm and we observed a flat ASE bandwidth from 1400 to 1570nm for the 5000ppm Tm3+ concentration.
Tellurite glasses optical fibers became promising for optical amplifiers due to its high rare earth ions solubility. One of the most important parameter for optical amplification is the lifetime of the excited states, which strongly depends on the crystal field around the ions. That crystal field changes dramatically with the host glass and with rare earth concentration. Excited state lifetime studies have been performed on Er3+-doped-TeO2-WO3-Na2O-Nb2O5 glass system. Therefore, we decided to study the Er3+ excited states lifetimes and compared with the Judd-Ofelt theory predictions. We measured only the 4I13/2 to 4I15/2 transitions lifetimes. The other transitions lifetimes can be inferred by knowing the Ω2, Ω4, Ω6 Judd-Ofelt parameters. These parameters were calculated with the electric-magnetic dipole transition oscillator strength for the desired excited levels and ground state obtained from the optical absorption spectra. After performing this calculation we estimated that maximum quantum efficiency (η), measured/calculated lifetimes ratio, would be achieved at 7500ppm (%wt) Er3+ content.
We produced a PbTe quantum dot core doped optical fiber with tellurite glasses intended to be used in highly nonlinear ultrafast optical devices capable to operate at the optical communication window at 1300 and 1500 nm wavelength region. Attenuation peaks of the optical fiber depends on the heat treatment time as expected for dots growth and covered the whole mid infrared region near 1500 nm. The optical fiber preform was made with the rod-in-tube method and the fiber was produced with a 4 m high Heatway drawing tower. The optical fiber core can be heavily doped because tellurite glasses solubility for PbTe quantum dots is order of magnitude higher than borosilicate and phosphate glasses, for example. In order to match all the requirements for core-clad optical fibers we studied undoped and doped tellurite glasses optical and thermophysical properties as a function of the glass composition. We also followed the growth kinetics of the quantum dots by High Resolution Transmission Electron Microscopy in the bulk glass matrix and the optical fiber.
We developed a set up consisting of an Optical Tweezers plus linear and non-linear micro-spectroscopy system to add the capabilities of manipulation and analysing the captured object. For the confocal micro-spectrometer we used a 30 cm monochromator equipped with a cooled back illuminated CCD. The spectroscopic laser system included a cw and a femtosecond Ti:sapphire lasers that allowed us to perfom raman, hyper-raman, hyper-rayleigh and two-photon excited (TPE) luminescence in trapped particles with an Nd:YAG cw laser. With the cw Ti:sapphire laser we obtained raman spectra of a single trapped polystyrene microsphere and red blood cells and silicon to evaluate the performance of our system. The femtosecond Ti:sapphire laser was used to observed hyper-rayleigh and hyper-raman peaks of SrTiO3 with 60s integration time only. In the past, hyper-raman measurements required integration times of few hours, but the huge intensity together with the 80 MHz repetition rate of the femtosecond laser decreased this time for the seconds range. The sensitiveness of our system also permitted to observe more than 14 Mie resonance peaks in the TPE luminescence of a single stained trapped microsphere, which agrees well with the calculations. This system opens up the possibility to perform spectroscopy in a living trapped micro-organism in any desired neighbourhood and dynamically observe the chemical reactions and/or mechanical properties change in real time.
In this work we used our set up consisting of an optical tweezers plus non-linear micro-spectroscopy system to perform scanning microscopy and observe spectra using two photon excited (TPE) luminescence of captured single cells conjugated with quantum dots of CdS and CdTe. The CdS nanocrystals are obtained by our group via colloidal synthesis in aqueous medium with final pH = 7 using sodium polyphosphate as the stabilizing agent. In a second step the surface of CdS particles is functionalized with linking agents such as Glutaraldehyde. The CdTe quantum dots are functionalized in the its proper synthesis using mercaptoacetic acid (AMA). We used a femtosecond Ti:sapphire laser to excite the hyper Rayleigh or TPE luminescence in particles trapped with an Nd:YAG cw laser and a 30 cm monochromator equipped with a cooled back illuminated CCD to select the spectral region for imaging. With this system we obtained hyper Rayleigh and TPE luminescence images of macrophages and other samples. The results obtained show the potential presented by this system and fluorescent labels to perform spectroscopy in a living trapped microorganism in any neighbourhood and dynamically observe the chemical reactions changes in real time.
The objective of this research is to use the displacements of a polystyrene microsphere trapped by an optical tweezers (OT) as a force transducer in mechanical measurements in life sciences. To do this we compared the theoretical optical and hydrodynamic models with experimental data under a broad variation of parameters such as fluid viscosity, refractive index, drag velocity and wall proximities. The laser power was measured after the objective with an integration sphere because normal power meters do not provide an accurate measurement for beam with high numerical apertures. With this careful laser power determination the plot of the optical force (calculated by the particle displacement) versus hydrodynamic force (calculated by the drag velocity) under very different conditions shows an almost 45 degrees straight line. This means that hydrodynamic models can be used to calibrate optical forces and vice-versa. With this calibration we observed the forces of polystyrene bead attached to the protozoa Leishmania amazonensis, responsible for a serious tropical disease. The force range is from 200 femto Newtons to 4 pico Newtons and these experiments shows that OT can be used for infection mechanism and chemotaxis studies in parasites. The other application was to use the optical force to measure viscosities of few microliters sample. Our result shows 5% accuracy measurements.
Optical tweezers (OT) rely on the radiation pressure to trap and manipulate microscopic particles and living microorganisms. Because the optical forces vary from hundreds of femto to tens of pico Newtons, OT can be used as an ultra sensitive force measurement tool to study interactions involving very small forces. We use a double tweezers to perform ultra sensitive measurement of the force due to the scattering of light as a function of its wavelength, in other words, to perform a Force Spectroscopy. Our results show not only the Mie resonances but also a selective coupling to either the TE, TM or both microsphere modes using the light polarization and the beam positioning. Mie resonances have usually been observed by scattering measurements. Very few reports of levitation experiments observed these resonances directly through the force. The double tweezers system has the advantage and flexibility of a stable restorative force measurement system. The experimental results show excellent agreement with Gaussian shaped beam partial wave decomposition theory. The understanding of the optical scattering forces in dielectric microspheres under different incident beam conditions is important as they have been used as the natural force transducer for mechanical measurements. Our results show how careful one has to be when using optical force models for this purpose. The Mie resonances can change the force values by 30-50%. Also the results clearly show how the usually assumed azimuthal symmetry in the horizontal plane no longer holds because the beam polarization breaks this symmetry.
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