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In the past decade, the field of biosensing has experienced an incredible pace of development, due to the compelling need for accurate and reliable tools for characterization of biomolecular kinetics. Specifically, label-free kinetic measurements are the most direct method for studying molecular binding, for example to establish the efficacy of drug-receptor interactions. For this reason, researchers in the pharmaceutical industry rely heavily on label-free detection for drug and antibody screening. Meanwhile, in the biosafety industry and healthcare, there is great demand for screening tools that can target biothreats, in order to accurately recognize the presence of toxins and pathogens with high sensitivity in diverse samples, such as bodily fluids, food and drinking water. This research topic has become particularly relevant during the recent pandemic, where vaccine development was carried out side by side with quantification and characterization of single viral particles. Here, we introduce a versatile biosensing platform capable of characterizing virtually any type of target compound, down to the single molecule level. For this work, we have improved the Interferometric Reflectance Imaging Sensor (IRIS) to perform accurate measurements of the binding kinetics of analytes ranging in molecular weight from less than 1kDa (small molecules) to more than 1MDa (biological nanoparticles). For the first time, we demonstrate multiplexed kinetic binding characterization of small molecules to surface immobilized antibody probes, as well as detection and phenotyping of large and complex analytes, on the same platform. The IRIS platform utilizes the optical interference signal produced by thinly layered substrates in order to precisely measure the thickness of a transparent film atop a silicon chip. In the context of this work, dynamic characterization of a wide range of biomolecular and nanoparticle targets was made possible by a multidimensional optimization, in order to improve both the sensitivity and the dynamic range of the instrument. Analysis of low molecular weight compounds required a significant increase in signal to noise ratio, which was achieved through averaging, as well as complete elimination of background solution effects ('bulk effect’). Additionally, the best surface chemistry for each application was identified by a new technique which consists of immobilizing capture probes on a multiplexed array of active polymers functionalized on the same sensor surface, allowing for simultaneous side-by-side comparison of their performance. Surface chemistry plays a huge role in kinetic measurements, in terms of probe functionality, steric hindrance, charge distribution and diffusion effects. Finally, imaging optics, illumination wavelength, and thickness of the silicon dioxide film were optimized to perform detection and phenotyping of large analytes, such as extracellular vesicles (EVs) and antibody-conjugated gold nanoparticles (mAb-GNPs). Results obtained from numerical simulations allowed for selection of the best experimental parameters for each application. Experimentally, mAb-GNPs were utilized to produce a real-time sandwich lateral flow assay. In this context, we demonstrated how the improved IRIS platform can bridge the gap between single-particle detection ('digital’ configuration) and bulk reflectance measurements ('analog’ configuration), creating a new 'hybrid' system (h-IRIS), which only requires minimal hardware adjustments to easily switch from one modality to the other. This brought a substantial improvement in sensitivity, improving the limit of detection by three orders of magnitude and enabling single-molecule level measurements. Finally, future system optimization ideas are presented to achieve even higher accuracy and further extend the range of target analytes.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Stimulated Raman spectroscopy (SRS) is a nondestructive tool for biochemical characterization of tissues. The aims were: 1) To evaluate the ability of SRS and Spontaneous Raman spectroscopy (SpRS) to differentiate among sound, demineralized and remineralized bovine enamel by phosphate and carbonate ratio (P/C-Ratio); and 2) To determine the correlation between the outcomes of transverse microradiography (TMR: Integrated mineral loss (ΔZ) and lesion depth) and P/C-Ratio. Material and Methods: Thirty, 5×5×2-mm ground and polished bovine enamel blocks were prepared. The surface was divided into 3 equal areas. Each area was chemically demineralized (demin) by Carbopol demineralized solution for 0 (Sound-Demin), 24 (24h-Demin), and 48h (48h-Demin), respectively. Then, specimens were sectioned for TMR analysis, and the remaining one part of each specimen was remineralized (remin) for 15days using a pH-cycling model (Sound-Remin, 24hD-Remin=24h-Demin and remineralization, 48hD-Remin = 48h-Demin and remineralization). Demin and remin groups were scanned to obtain P/C-Ratio by SpRS and SRS. SRS was further scanned from 0 (surface) up to 100 µm into the dentine at 10-µm intervals. Remineralized specimens were sectioned for TMR analysis. Wilcoxon signed-rank tests were used to compare between TMR and SpRS/SRS. Spearman correlation coefficients were used to correlate among TMR, SpRS, and SRS. A 5-percent significance level was used for each test. Results: As demin time increased, both ΔZ and lesion depth were increased. After remineralization, both values were decreased. There were significant differences between demine and remin groups and between demin times. For SpRS, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin and 48h-Demin (p ≤ 0.001). The 24h-Demin had significantly larger values than 48h-Demin (p = 0.048). Sound-Remin had larger P/C-Ratio than 24hD-Remin (p = 0.316) and 48hD-Remin (p = 0.015). 24hD-Remin was larger than 48hD-Remin (p = 0.269). 24hD-Remin had significantly larger P/C-Ratio than 24h-Demin (p ≤ 0.001). 48hD-Remin had significantly larger P/C-Ratio than 48h-Demin (p ≤ 0.001). For SRS, at surface (0 µm), for demin group, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin (p = 0.020) and 48h-Demin (p = 0.032). 24h-Demin had larger value than 48h-Demin; but no significant difference (p = 0.117). Among remin groups, Sound-Remin was not statistical significance different for 24hD-Remin (p = 0.172) and 48hD-Remin (p = 0.134). However, 24hD-Remin was smaller; but not statistical significance different from 48hD-Remin (p = 0.688). At deeper levels (10 µm to 100µm), it was found that 1) After demineralization, Sound-Demin had significantly larger P/C-Ratio than 24h-Demin and 48h-Demin at 0 µm to 20 µm, and 80 µm to 100µm; Sound-Demin had significantly larger P/C-Ratio than 48h-Demin; and no statistical significance differences were found among Sound-Demin and 24h-Demin, 24h-Demin and 48h-Demin. 2) After remineralization, no statistical significance differences were found among Sound-Remin, 24hD-Remin, and 48hD-Remin. 3) Sound-Demin had significantly larger P/C-Ratio than Sound-Remin at 0 µm ,10 µm, 20 µm; and no statistical significance differences were found at levels deeper than 30 µm. 4) No statistical significance differences were found between 24h-Demin and 24hD-Remin from 0 µm to 70µm; and 24hD-Remin had significantly larger P/C-Ratio than 24h-Demin from 80 µm to 100 µm. 5) No statistical significance differences were found between 48h-Demin and 48hD-Remin. For correlation, moderate correlation was found between SpRS demineralized/remineralized groups and ΔZ, and between SpRS demineralized groups and lesion depth. Conclusion: SpRS and SRS have the potential to quantify demineralization through calculation of the phosphate and carbonate ratio. In addition, SpRS can detect the change of remineralization. A nondestructive caries detection approach using SpRS and SRS would be beneficial in clinical practice.

No description available.

Fluorescence microscopy is an indispensable tool in the areas of cell biology, histology and material science as it enables non-invasive observation of specimen in their natural environment. The main advantage of fluorescence microscopy is that, it is non-invasive and capable of imaging with very high contrast and visibility. It is dynamic, sensitive and allows high selectivity. The specificity and sensitivity of antibody-conjugated probes and genetically-engineered fluorescent protein constructs allows the user to label multiple targets and the precise location of intracellular components. However, its spatial reso- lution is limited to one-quarter of the excitation wavelength (Abbe’s diffraction limit). The advent of new and sophisticated optics and availability of fluorophores has made fluorescence imaging a flourishing field. Several advanced techniques like TIRF, 4PI, STED, SIM, SPIM, PALM, fPALM, GSDIM and STORM, have enabled high resolution imaging by breaking the diffraction barrier and are a boon to medical and biological research. Invention of confocal and multi-photon microscopes have enabled observation of the specimen embedded at depth. All these advances in fluorescence microscopy have made it a much sought-after technique. The first chapter provides an overview of the fundamental concepts in fluorescence imag- ing. A brief history of emergence of the field is provided in this chapter along with the evolution of different super-resolution microscopes. An introduction to the concept of fluorophores, their broad classification and their characteristics is discussed in this chap- ter. A brief explanation of different fluorescence imaging techniques and some trending techniques are introduced. This chapter provides a thorough foundation for the research work presented in the thesis. Second chapter deals with different microscopy techniques that have changed the face of biophotonics and nanoscale imaging. The resolution of optical imaging systems are dictated by the inherent property of the system, known as impulse response or more popularly “point spread function”. A basic fluorescence imaging system is presented in this chapter and introduces the concept of point spread function and resolution. The introduction of confocal microscope and multi-photon microscope brought about improved optical sectioning. 4PI microscopy technique was invented to improve the axial resolution of the optical imaging system. Using this microscopy modality, an axial resolution of upto ≈ 100nm was made possible. The basic concepts of these techniques is provided in this chapter. The chapter concludes with a discussion on some of the optical engineering techniques that aid in improved lateral and axial resolution improvements and then we proceed to take on these engineering techniques in detail in the next chapter. Introduction of spatial masks at the back aperture of the objective lens results in gen- eration of a Bessel-like beam, which enhances our ability to see deeper inside a spec- imen with reduced aberrations and improved lateral resolution. Bessel beams have non-diffracting and self-reconstructing properties which reduces the scattering while ob- serving cells embedded deep in a thick tissue. By coupling this with the 4PI super- resolution microscopy technique, multiple excitation spots can be generated along the optical axis of the two opposing high-NA objective lenses. This technique is known as multiple excitation spot optical (MESO) microscopy technique. It provides a lateral resolution improvement upto 150nm. A detailed description of the technique and a thorough analysis of the polarization properties is discussed in chapter 3. Chapters 4 and 5 bring the focus of the thesis to the main topic of research - multi- dimensional image reconstruction for fluorescence microscopy by employing the statis- tical techniques. We begin with an introduction to filtering techniques in Chapter 4 and concentrate on an edge-preserving denoising filter: Bilateral Filter for fluorescence microscopy images. Bilateral filter is a non-linear combination of two Gaussian filters, one based on proximity of two pixels and the other based on the intensity similarity of the two. These two sub-filters result in the edge-preserving capability of the filter. This technique is very popular in the field of image processing and we demonstrate the application of the technique for fluorescence microscopy images. The chapter presents a through description of the technique along with comparisons with Poisson noise mod- eling. Chapters 4 and 5 provide a detailed introduction to statistical iterative recon- struction algorithms like expectation maximization-maximum likelihood (EM-ML) and maximum a-posteriori (MAP) techniques. The main objective of an image reconstruc- tion algorithm is to recover an object from its noisy degraded images. Deconvolution methods are generally used to denoise and recover the true object. The choice of an appropriate prior function is the crux of the MAP algorithm. The remaining of chapter 5 provides an introduction to different potential functions. We show some results of the MAP algorithm in comparison with that of ML algorithm. In chapter 6, we continue the discussion on MAP reconstruction where two new potential functions are introduced and demonstrated. The first one is based on the application of Taylor series expansion on the image. The image field is considered to be analytic and hence Taylor series produces an accurate estimation of the field being reconstructed. The second half of the chapter introduces an interpolation function to approximate the value of a pixel in its neighborhood. Cubic B-splines are widely used as a basis function during interpolation and they are popular technique in computer vision and medical imaging techniques. These novel algorithms are tested on di_erent microscopy data like, confocal and 4PI. The results are shown at the _nal part of the chapter. Tagging cell organelles with uorescent probes enable their visualization and analysis non-invasively. In recent times, it is common to tag more than one organelle of interest and simultaneously observe their structures and functions. Multicolor uorescence imaging has become a key technique to study speci_c processes like pH sensing and cell metabolism with a nanoscale precision. However, this process is hindered by various problems like optical artifacts, noise, autouorescence, photobleaching and leakage of uorescence from one channel to the other. Chapter 7 deals with an image reconstruction technique to obtain noise-free and distortion-less data from multiple channels when imaging a multicolor sample. This technique is easily adaptable with the existing imaging systems and has potential application in biological imaging and biophysics where multiple probes are used to tag the features of interest. The fact that the lateral resolution of an optical system is better than the axial resolution is well known. Conventional microscopes focus on cells that are very close to the cover-slip or a few microns into the specimen. However, for cells that are embedded deep in a thick sample (ex: tissues), it is di_cult to visualize them using a conventional microscope. A number of factors like, scattering, optical aberrations, mismatch of refractive index between the objective lens and the mounting medium and noise, cause distortion of the images of samples at large depths. The system PSF gets distorted due to di_raction and its shape changes rapidly at large depths. The aim of chapter 8 is to introduce a technique to reduce distortion of images acquired at depth by employing image reconstruction techniques. The key to this methodology is the modeling of PSF at large depths. Maximum likelihood technique is then employed to reduce the streaking e_ects of the PSF and removes noise from raw images. This technique enables the visualization of cells embedded at a depth of 150_m. Several biological processes within the cell occur at a rate faster than the rate of acquisition and hence vital information is missed during imaging. The recorded images of these dynamic events are corrupted by motion blur, noise and other optical aberrations. Chapter 9 deals with two techniques that address temporal resolution improvement of the uorescence imaging system. The _rst technique focuses on accelerating the data acquisition process. This includes employing the concept of time-multiplexing to acquire sequential images from a dynamic sample using two cameras and generating multiple sheets of light using a di_raction grating, resulting in multi-plane illumination. The second technique involves the use of parallel processing units to enable real-time image reconstruction of the acquired data. A multi-node GPU and CUDA architecture effciently reduce the computation time of the reconstruction algorithms. Faster implementation of iterative image reconstruction techniques can aid in low-light imaging and dynamic monitoring of rapidly moving samples in real time. Employing rapid acquisition and rapid image reconstruction aids in real-time visualization of cells and have immense potential in the _eld of microbiology and bio-mechanics. Finally, we conclude the thesis with a brief section on the contribution of the thesis and the future scope the work presented. Thank you for using www.freepdfconvert.com service! 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<p>Altered metabolism is a hallmark of almost all cancers. A tumor’s metabolic phenotype can drastically change its ability to proliferate and to survive stressors such as hypoxia or therapy. Metabolism can be used as a diagnostic marker, by differentiating neoplastic and normal tissue, and as a prognostic marker, by providing information about tumor metastatic potential. Metabolism can further be used to guide treatment selection and monitoring, as cancer treatments can influence metabolism directly by targeting a specific metabolic dysfunction or indirectly by altering upstream signaling pathways. Repeated measurement of metabolic changes during the course of treatment can therefore indicate a tumor’s response or resistance. Recently, well-supported theories indicate that the ability to modulate metabolic phenotype underpins some cancer cells’ ability to remain dormant for decades and recur with an aggressive phenotype. It follows that accurate identification and repeated monitoring of a tumor’s metabolic phenotype can bolster understanding and prediction of a tumor’s behavior from diagnosis, through treatment, and (sadly) sometimes back again.</p><p>The two primary axes of metabolism are glycolysis and mitochondrial metabolism (OXPHOS), and alteration of either can promote unwanted outcomes in cancer. In particular, increased glucose uptake independent of oxygenation is a well-known mark of aggressive cancers that are more likely to metastasize and evade certain therapies. Lately, mitochondria are also gaining recognition as key contributors in tumor metabolism, and mitochondrial metabolism has been shown to promote metastasis in a variety of cell types. Most tumor types rely on a combination of both aerobic glycolysis and mitochondrial metabolism, but the two axes’ relative contributions to ATP production can vary wildly. Knowledge of both glycolytic and mitochondrial endpoints is required for actionable, systems-level understanding of tumor metabolic preference. </p><p>Several technologies exist that can measure endpoints informing on glycolytic and/or mitochondrial metabolism. However, these technologies suffer from a combination of prohibitive cost, low resolution, and lack of repeatability due to destructive sample treatments.</p><p>There is a critical need to bridge the gap in pre-clinical studies between single-endpoint whole body imaging and destructive ex vivo assays that provide multiple metabolic properties, neither of which can provide adequate spatiotemporal information for repeated tumor monitoring. Optical technologies are well-suited to non-destructive, high resolution imaging of tumor metabolism. A carefully chosen set of endpoints can be measured across a variety of length scales and resolutions to obtain a complete picture of a tumor’s metabolic state. First, the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) can be used to report on glucose uptake. The fluorophore tetramethylrhodamine, ethyl ester (TMRE) reports on mitochondrial membrane potential, which provides information regarding capacity for oxidative phosphorylation. Vascular oxygenation (SO2) and morphological features, which are critical for interpretation of 2-NBDG and TMRE uptake, can be obtained using only endogenous contrast from hemoglobin. </p><p>Three specific aims were proposed toward the ultimate goal of developing an optical imaging toolbox that utilizes exogenous fluorescence and endogenous absorption contrast to characterize cancer metabolic phenotype in vivo. </p><p>In Aim 1, we optimized the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) to report on glycolytic demand in vivo. Our primary goal was to demonstrate that correcting 2-NBDG uptake (NBDG60) by the rate of delivery (RD) showed improved contrast between distinct tumor phenotypes. We showed that the ratio 2-NBDG60/RD served as a delivery-corrected measure of glucose uptake in the dorsal skin flap window chamber models containing normal tissues and tumors. Delivery correction was able to minimize the effects of a large change in the injected 2-NBDG dose. Further, the endpoint showed a significant inverse correlation with blood glucose levels. Since glucose has been shown to competitively inhibit 2-NBDG transport into cells, this finding indicating that we were indeed reporting on glucose uptake. Importantly, the ratio was able to distinguish specific uptake of 2-NBDG from accumulation of a fluorescent control, 2-NBDLG, which is identical to 2-NBDG in molecular weight and fluorescent spectrum, but is unable to undergo active transport into the cell. </p><p>The ratio 2-NBDG60/RD was then leveraged to compare different tumor phenotypes and to characterize the dependence of glucose uptake on vascular oxygenation within these tumors. Our results showed that 2-NBDG60/RD was an effective endpoint for comparing in vivo glucose uptake of metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. Further, the addition of vascular information revealed metabolic heterogeneity within the tumors. The primary conclusion of Aim 1 was that delivery-corrected 2-NBDG uptake (2-NBDG60/RD) is an appropriate indicator of glucose demand in both normal and tumor tissues.</p><p>In Aim 2, we optimized fluorescent tetramethyl rhodamine, ethyl ester (TMRE) for measurement of mitochondrial membrane potential (MMP). We then leveraged the relationships between MMP, glucose uptake, and vascular endpoints to characterize the in vivo metabolic landscapes of three distinct and extensively studied murine breast cancer lines: metastatic 4T1 and non-metastatic 67NR and 4T07. </p><p>Using two-photon microscopy, we confirmed that TMRE localizes to mitochondrial-sized features in the window chamber when delivered via tail vein. The kinetics of TMRE uptake were robust across both normal and tumor tissues, with a stable temporal window for measurement from 40-75 minutes after injection. We saw that TMRE uptake decreased as expected in response to hypoxia in non-tumor tissue, and in response to chemical inhibition with a mitochondrial uncoupler in both non-tumor and 4T1 tissue. MMP was increased in all tumor types relative to non-tumor (p<0.05), giving further confirmation that TMRE was reporting on mitochondrial activity.</p><p>We leveraged the relationships between the now-optimized endpoints of MMP (Aim 2), glucose uptake (Aim 1) and vascular endpoints (Aims 1 and 2) to characterize the in vivo metabolic landscapes of three distinct and extensively studied murine breast cancer lines: metastatic 4T1 and non-metastatic 67NR and 4T07. Imaging the combination of endpoints revealed a classic “Warburg effect” coupled with hyperpolarized mitochondria in 4T1; 4T1 maintained vastly increased glucose uptake and comparable MMP relative to 4T07 or 67NR across all SO2. We also showed that imaging trends were concordant with independent metabolomics analysis, though the lack of spatial and vascular data from metabolomics obscured a more detailed comparison of the technologies.</p><p>We observed that vascular features in tumor peritumoral areas (PA) were equally or more aberrant than vessels in the tumor regions that they neighbored. This prompted consideration of the metabolic phenotype of the PA. Regional metabolic cooperation between the tumor region and the PA was seen only in 4T1, where MMP was greater in 4T1 tumors and glucose uptake was greater in 4T1 PAs. </p><p>Because of their regional metabolic coupling as well as their demonstrated capacity for glycolysis and mitochondrial activity, we hypothesized that the 4T1 tumors would have an increased ability to maintain robust MMP during hypoxia. 67NR and 4T07 tumors showed expected shifts toward decreased MMP and increased glucose uptake during hypoxia, similar to the trends we observed in normal tissue. Surprisingly, 4T1 tumors appeared to increase mitochondrial metabolism during hypoxia, since MMP increased and SO2 dramatically decreased. Overall, this aim demonstrated two key findings: 1. TMRE is a suitable marker of mitochondrial membrane potential in vivo in normal tissue and tumors, and 2. imaging of multiple metabolic and vascular endpoints is crucial for the appropriate interpretation of a metabolic behavior. </p><p>Finally, in Aim 3 we evaluated the feasibility of combined 2-NBDG and TMRE imaging. The primary objective was to enable simultaneous imaging of the two fluorophores by minimizing sources of “cross-talk”: chemical reaction, optical overlap, and confounding biological effects. A secondary objective was to transition our imaging method to a new platform, a reflectance-mode, high-resolution fluorescence imaging system built in our lab, which would expand the use of our technique beyond the dorsal window chamber model. We first used liquid chromatography- mass spectrometry to confirm that the chemical properties of the two fluorophores were compatible for simultaneous use, and indeed saw that the mixing of equimolar 2-NBDG and TMRE did not form any new chemical species. </p><p>We also performed a phantom study on the hyperspectral imaging system, used for all animal imaging in Aim 1 and Aim 2, to estimate the range of 2-NBDG and TMRE concentrations that are seen at the tissue level in normal and tumor window chambers. We created a new phantom set that spanned the range of estimated in vivo concentrations, and imaged them with the reflectance-mode fluorescence imaging system. The phantom experiments gave us two important findings. First, we saw that fluorescence intensity increased linearly with fluorophore concentration, allowing for accurate quantification of concentration changes between samples. Most importantly, we found that we could exploit the optical properties of the fluorophores and our system’s spectral detection capability to excite the two fluorophores independently. Specifically, we could excite 2-NBDG with a 488nm laser without detectable emission from TMRE, and could excite TMRE with a 555nm laser without detectable emission from 2-NBDG. With this characterization, the optical properties of the two fluorophores were considered compatible for simultaneous imaging. </p><p>Next, we sought to determine whether biological or delivery interactions would affect uptake of the two fluorophores. Surprisingly, both in vitro and in vivo imaging suggested that simultaneous dosing of the 2-NBDG and TMRE caused significant changes in uptake of both probes. Since we previously found that TMRE equilibrates rapidly at the tissue site, we hypothesized that staggering the injections to allow delivery of TMRE to tissue before injecting 2-NBDG would restore the full uptake of both fluorophores. Two sequential injection protocols were used: in the first group, TMRE was injected first followed by injection of 2-NBDG after only 1-5 minutes, and in the second group, TMRE was injected first followed by injection of 2-NBDG after 10-15 minutes. Both sequential injection strategies were sufficient to restore the final fluorescence of both fluorophores to that seen in the separate TMRE or 2-NBDG imaging cohorts; however, the shorter time delay caused changes to the initial delivery kinetics of 2-NBDG. We concluded that sequential imaging of TMRE followed by 2-NBDG with a 10-15 minute delay was therefore the optimal imaging strategy to enable simultaneous quantification of glucose uptake and mitochondrial membrane potential in vivo. </p><p>Applying the sequential imaging protocol to 4T1 tumors demonstrated a highly glycolytic phenotype compared to the normal animals, as we had seen in Aim 2. However, mitochondrial membrane potential was comparable for the normal and tumor groups. The next study will test an extended delay between the injections to allow more time for TMRE delivery to tumors prior to 2-NBDG injection. Overall, the key finding of Aim 3 was that a carefully chosen delivery strategy for 2-NBDG and TMRE enabled simultaneous imaging of the two endpoints, since chemical and optical cross-talk were negligible.</p><p>The work presented here indicates that an optical toolbox of 2-NBDG, TMRE, and vascular endpoints is well poised to reveal interesting and distinct metabolic phenomena in normal tissue and tumors. Future work will focus on the integration of optical spectroscopy with the microscopy toolbox presented here, to enable long-term studies of the unknown metabolic changes underlying a tumor’s response to therapy, its escape into dormancy, and ultimately, its recurrence.</p> / Dissertation

A limpeza e modelagem dos canais radiculares consiste em uma etapa fundamental para o sucesso da terapia endodôntica. O objetivo deste estudo foi avaliar um novo conceito de instrumento endodôntico Self Adjusting File (SAF), e instrumentos rotatórios de NiTi, na capacidade de limpeza de canais achatados por meio de microscopia óptica. Vinte e dois incisivos inferiores instrumentados com instrumentos rotatórios K3 (n=11) e sistema SAF (n=11), tiveram seus terços apicais submetidos ao processamento histológico e analisados em microscopia óptica (40×). As imagens capturadas pelo software Adobe Photoshop 5.1 foram analisadas com auxilio de grade de integração do software Image J. O perímetro de ação dos instrumentos nas paredes dos canais radiculares foi determinado com auxilio do software Image J e uma mesa digital Bamboo. Os dados obtidos foram submetidos à análise de variância e teste de T não pareado corrigido pelo teste de Welch, considerando-se a presença de debris no terço apical e a superfície de dentina sem contato com o instrumento (&alpha;=5%). A análise estatística mostrou haver diferença significante entre os grupos. Canais instrumentados com o sistema endodôntico SAF apresentaram valores percentual de debris e superfície não tocada pelo instrumento menores (2,18 ± 2,71 e 12,33 ± 7,85 respectivamente) quando comparado com canais instrumentados com instrumentos rotatórios (13,11 ± 12,98 e 53,54 ± 15,95, respectivamente) (p<0.05). Concluiu-se que o sistema SAF proporcionou melhor preparo de canais radiculares por tocar mais nas paredes radiculares promovendo consequentemente melhor limpeza. / Cleaning and shaping of root canals are essential steps for the success of endodontic therapy. The purpose of this study was to evaluate a new concept of endodontic instrument - the Self Adjusting File (SAF) system - and NiTi rotary instruments, regarding their cleaning capacity in flattened root canals, using optical microscopy. Twenty-two mandibular incisors prepared with K3 rotary instruments (n=11) and SAF system (n=11) had their apical thirds subjected to histological processing and analyzed by optical microscopy (40×). The images captured by Adobe Photoshop 5.1 software were analyzed with the integration grid of Image J software. The perimeter of action of the instruments on the root canals walls was determined using Image J software and a Bamboo digital tablet. Data were analyzed statistically using analysis of variance and unpaired t-test with Welchs correction, considering the presence of debris in the apical third and the root dentin surface untouched by the instrument (&alpha;=5%). The statistical analysis revealed significant difference between the groups. Canals prepared with the SAF system presented lower percent values of debris and untouched root dentin surfaces (2.18 ± 2.71 and 12.33 ± 7.85, respectively) compared with canals prepared with rotary instruments (13.11 ± 12.98 and 53.54 ± 15.95, respectively) (p<0.05). In conclusion, the SAF system was associated with better root canal preparation, as it touched more the root dentin walls and consequently promoted a more effective cleaning.

A limpeza e modelagem dos canais radiculares consiste em uma etapa fundamental para o sucesso da terapia endodôntica. O objetivo deste estudo foi avaliar um novo conceito de instrumento endodôntico Self Adjusting File (SAF), e instrumentos rotatórios de NiTi, na capacidade de limpeza de canais achatados por meio de microscopia óptica. Vinte e dois incisivos inferiores instrumentados com instrumentos rotatórios K3 (n=11) e sistema SAF (n=11), tiveram seus terços apicais submetidos ao processamento histológico e analisados em microscopia óptica (40×). As imagens capturadas pelo software Adobe Photoshop 5.1 foram analisadas com auxilio de grade de integração do software Image J. O perímetro de ação dos instrumentos nas paredes dos canais radiculares foi determinado com auxilio do software Image J e uma mesa digital Bamboo. Os dados obtidos foram submetidos à análise de variância e teste de T não pareado corrigido pelo teste de Welch, considerando-se a presença de debris no terço apical e a superfície de dentina sem contato com o instrumento (&alpha;=5%). A análise estatística mostrou haver diferença significante entre os grupos. Canais instrumentados com o sistema endodôntico SAF apresentaram valores percentual de debris e superfície não tocada pelo instrumento menores (2,18 ± 2,71 e 12,33 ± 7,85 respectivamente) quando comparado com canais instrumentados com instrumentos rotatórios (13,11 ± 12,98 e 53,54 ± 15,95, respectivamente) (p<0.05). Concluiu-se que o sistema SAF proporcionou melhor preparo de canais radiculares por tocar mais nas paredes radiculares promovendo consequentemente melhor limpeza. / Cleaning and shaping of root canals are essential steps for the success of endodontic therapy. The purpose of this study was to evaluate a new concept of endodontic instrument - the Self Adjusting File (SAF) system - and NiTi rotary instruments, regarding their cleaning capacity in flattened root canals, using optical microscopy. Twenty-two mandibular incisors prepared with K3 rotary instruments (n=11) and SAF system (n=11) had their apical thirds subjected to histological processing and analyzed by optical microscopy (40×). The images captured by Adobe Photoshop 5.1 software were analyzed with the integration grid of Image J software. The perimeter of action of the instruments on the root canals walls was determined using Image J software and a Bamboo digital tablet. Data were analyzed statistically using analysis of variance and unpaired t-test with Welchs correction, considering the presence of debris in the apical third and the root dentin surface untouched by the instrument (&alpha;=5%). The statistical analysis revealed significant difference between the groups. Canals prepared with the SAF system presented lower percent values of debris and untouched root dentin surfaces (2.18 ± 2.71 and 12.33 ± 7.85, respectively) compared with canals prepared with rotary instruments (13.11 ± 12.98 and 53.54 ± 15.95, respectively) (p<0.05). In conclusion, the SAF system was associated with better root canal preparation, as it touched more the root dentin walls and consequently promoted a more effective cleaning.

No description available.