A. A. de Thomaz

Studying taxis in real time using optical tweezers: Applications for Leishmania amazonensis parasites

Beads trapped by an optical tweezers can be used as a force transducer for measuring forces of the same order of magnitude as typical forces induced by flagellar motion. We used an optical tweezers to study chemotaxis by observing the force response of a flagellated microorganism when placed in a gradient of attractive chemical substances. This report shows such observations for Leishmania amazonensis, responsible for leishmaniasis, a serious disease. We quantified the movement of this protozoan for different gradients of glucose. We were able to observe both the strength and the directionality of the force. The characterization of the chemotaxis of these parasites can help to understand the mechanics of infection and improve the treatments employed for this disease. This methodology can be used to quantitatively study the taxis of any kind of flagellated microorganisms under concentration gradients of different chemical substances, or even other types of variable gradients such as temperature and pressure.

Double optical tweezers for ultrasensitive force spectroscopy in microsphere Mie scattering

We used a double tweezers setup to perform ultrasensitive force spectroscopy and observe the forces due to light scattering in a single isolated particle. We demonstrate how to selectively couple the light to the transverse electric (TE), transverse magnetic (TM), or both TE and TM microsphere modes by means of the beam polarization and positioning, and to observe correspondent morphology-dependent resonances (MDR). The results show how the usually assumed azimuthal symmetry in the horizontal plane no longer holds because of the symmetry break caused by the beam polarization. Also, the MDR resonances can change the force values by more than 30–50%.

Use of the second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

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.

Optical tweezers for studying taxis in parasites

In this work we present a methodology to measure force strengths and directions of living parasites with an optical tweezers setup. These measurements were used to study the parasites chemotaxis in real time. We observed behavior and measured the force of: (i) Leishmania amazonensis in the presence of two glucose gradients; (ii) Trypanosoma cruzi in the vicinity of the digestive system walls, and (iii) Trypanosoma rangeli in the vicinity of salivary glands as a function of distance. Our results clearly show a chemotactic behavior in every case. This methodology can be used to study any type of taxis, such as chemotaxis, osmotaxis, thermotaxis, phototaxis, of any kind of living microorganisms. These studies can help us to understand the microorganism sensory systems and their response function to these gradients.

The role of stress in CdTe quantum dot doped glasses

In this work, we unequivocally demonstrate the influence of matrix-related stresses on quantum dots by measuring, side by side, a CdTe quantum dot doped glass and a colloidal sample with similar sizes. We measured the fluorescence spectra and fluorescence lifetime for both samples as a function of the temperature. We show that the expansion coefficient mismatch between CdTe quantum dots and the glass host causes stresses and drastically changes its behavior compared to its colloidal counterpart, even leading to phase transitions. This finding indicates that most experimental data on glass-doped quantum dots used to validate confinement models should be revised, taking stress into account.

Elastic fibers and collagen distribution in human aorta

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.

Multimodal nonlinear optical microscopy used to discriminate epithelial 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.

Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

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.

Combined nonlinear laser imaging (two-photon excitation fluorescence, second and third-harmonic generation, and fluorescence lifetime imaging microscopies) in ovarian tumors

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.

Quantitative second-harmonic generation imaging to detect osteogenesis imperfecta in human skin samples

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.