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.
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.
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.
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.
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.
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.
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.
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.
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.
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 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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