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.
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.
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 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.
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.
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.
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