Spectroscopic Insight into Oxidative Heme Cleavage by the Non-canonical Heme Oxygenase IsdG from Staphylococcus aureus

IsdG and IsdI are non-canonical heme oxygenases (HO) from Staphylococcus aureus that catalyze the oxidative cleavage of heme to give novel organic products (staphylobilins) and iron as a nutrient for the pathogen. Comparison of the reported equilibrium dissociation constant (Kd) values for heme from IsdG and IsdI compared to the reported concentration of the labile heme pool called into question whether these enzymes are competent HOs in vivo. We took advantage of a second-sphere Trp whose fluorescence is quenched upon heme binding, which led to Kd values 2-3 orders of magnitude smaller than reported in the literature. Importantly, these Kd values were on the same order of magnitude as human HO, precluding design of a competitive inhibitor as an effective therapeutic. Based upon the kinetic and equilibrium data, and the finding that the half-life of IsdG is increased 2.5-fold by the presence of heme, we proposed IsdG is the main HO involved in iron acquisition which motivated further characterization of IsdG. IsdG-catalyzed heme catabolism proceeds through ferric-peroxoheme and meso-hydroxyheme intermediates en route to staphylobilin. A second-sphere Asn is known to be critical for enzymatic function, but its role in heme cleavage was unknown. Site-directed mutagenesis was employed to probe the role of Asn using ferric-azidoheme and ferric-cyanoheme as models of the putative ferric-peroxoheme intermediate. An optical spectroscopic study established that a hydrogen-bond between Asn and the iron-ligating (α) atom of the distal ligand perturbs the heme electronic structure. Density functional theory (DFT) suggested this hydrogen-bond triggers rotation of the distal ligand, which was corroborated by circular dichroism (CD), and delocalizes spin density onto the meso carbons. Electron paramagnetic resonance (EPR) revealed the Asn hydrogen-bond increases the Fe 3dxy character in the singly occupied molecular orbital (SOMO), a mechanism that can increase spin density on the meso carbons. Finally, the Asn hydrogen-bond moves the meso carbon resonances downfield in the 13C nuclear magnetic resonance (NMR) spectrum, consistent with excess spin density, confirming a DFT-predicted, Asn-induced spin delocalization. These results suggest IsdG funnels the reactivity of ferric-peroxoheme toward heme hydroxylation through an Asn-dependent bridged transition state, circumventing production of reactive, uncontrolled intermediates

High Pressure Micro-Spectroscopy of Biological Assemblies and Cells

Functional properties of living cells depend on the thermodynamic variables such as temperature and pressure. A unique tool to investigate volume effects on structure and metabolism of the cell and biomolecules is pressure perturbation. We have developed a new setup that enables micro-spectroscopy and optical imaging of individual live cells at variable pressure from 0.1 to 400 MPa. Following characterization of the setup, pressure and temperature effects on the secondary structure of the peptide Poly-L-glutamic acid (PGA) in deuterated water buffer solution were investigated. The amide I band of PGA is sensitive to pressure and temperature, and by spectral deconvolution, we determined the relative contributions due to the ?-helix and random coil conformations. The population of ?-helix increases with increasing pressure. Pressure effects on single red blood cells and the intracellular protein hemoglobin were studied by micro-Raman spectroscopy. In particular, we observed a shift in the frequency of the iron-histidine vibrational band in both the intracellular hemoglobin and hemoglobin in solutions. The iron-histidine mode is a sensitive structural marker of the crucial iron-protein linkage in heme proteins. The pressure dependent shift suggests a conformational change of the heme environment. This finding was further supported by micro-absorption measurements at variable pressure. In additional experiments, Raman spectroscopy was employed to probe molecular changes that occurred in hemoglobin in erythrocytes infected with the malaria parasite Plasmodium falciparum. The spectra of infected cells indicated that hemoglobin degradation can be correlated with the stages of the parasite multiplication cycle. The research was further extended towards probing size and shape changes of individual cells with pressure. The lateral diameter in yeast cells was observed to decrease with pressure in a reversible way. These results suggest that transport of the intra-cellular water may play a significant role for volume changes. In summary, pressure changes were shown to induce conformational changes in proteins and shape changes in yeast cells. A Raman technique was developed to monitor the states of Plasmodium falciparum multiplication cycle within a red blood cell

Polarization-sensitive Mueller-matrix optical coherence tomography

Measuring the Mueller matrix with optical coherence tomography (OCT) makes it possible to acquire the complete polarization properties of scattering media with three-dimensional spatial resolution. We first proved that the measured degree-of-polarization (DOP) of the backscattered light by OCT remains unity-a conclusion that validated the use of Jones calculus in OCT. A multi-channel Mueller-matrix OCT system was then built to measure the Jones-matrix, which can be transformed into a Mueller matrix, images of scattering biological tissues accurately with single depth scan. We showed that when diattenuation is negligible, the round-trip Jones matrix represents a linear retarder, which is the foundation of conventional PS-OCT, and can be calculated with a single incident polarization state although the one-way Jones matrix generally represents an elliptical retarder; otherwise, two incident polarization states are needed. We discovered the transpose symmetry in the roundtrip Jones matrix, which is critical for eliminating the arbitrary phase difference between the two measured Jones vectors corresponding to the two incident polarization states to yield the correct Jones matrix. We investigated the various contrast mechanisms provided by Mueller-matrix OCT. Our OCT system for the first time offers simultaneously comprehensive polarization contrast mechanisms including the amplitude of birefringence, the orientation of birefringence, and the diattenuation in addition to the polarization-independent intensity contrast, all of which can be extracted from the measured Jones or the equivalent Mueller matrix. The experimental results obtained from rat skin samples, show that Mueller OCT provides complementary structural and functional information on biological samples and reveal that polarization contrast is more sensitive to thermal degeneration of biological tissues than amplitude-based contrast. Finally, an optical-fiber-based multi-channel Mueller-matrix OCT was built and a new rigorous algorithm was developed to retrieve the calibrated polarization properties of a sample. For the first time to our knowledge, fiber-based polarization-sensitive OCT was dynamically calibrated to eliminate the polarization distortion caused by the single-mode optical fiber in the sample arm, thereby overcoming a key technical impediment to the application of optical fibers in this technology

Development of an innovative real-time assay for antimalarial sensitivity testing

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2017Antimalarial drug resistance has always been an obstacle in the fight against malaria. Malaria parasites have developed resistance to most available antimalarial drugs and, more recently resistance to artemisinins, the first-line treatment for malaria, is emerging and spreading in Southeast Asia. Artemisinin resistance is characterized by delayed parasite clearance times observed in malaria patients. For now this resistance is considered to be partial by the WHO and artemisinin combination therapies remain as the mainstay of antimalarial treatment. In vitro assays are of paramount importance to detect and monitor drug resistance. Several in vitro drug assays exist, however their inherent characteristics, such as the use of radioactive or expensive reagents and their long turn-around times limit their application. The project developed in the context of this thesis aimed to develop a novel drug assay for Plasmodium spp. that would overcome some of the limitations of currently available drug assays. The underlying idea was to use hemozoin, a crystal produced by malaria parasites, to measure their own growth or maturation which would allow to detect drug effects. The hemozoin content increases as parasites mature inside the erythrocytes. Thus, hemozoin constitutes an optimal parasite maturation biomarker. Moreover, it is a birefringent crystal, and as such it is able to depolarize light. The resulting light depolarization can be easily detected by optical methods such as flow cytometry. It was previously shown by our laboratory that in a rodent model of malaria depolarization caused by hemozoin inside infected erythrocytes could be detected using a simple flow cytometric set-up. Moreover, the inhibitory effect of commonly used antimalarial drugs could also be determined after only 6-8 hours of incubation. The main objectives of the work developed during this thesis was to further develop the flow cytometric detection of hemozoin assay using P. falciparum in vitro cultures and to assess its performance ex vivo, in the field, using samples from malaria patients. A benchtop flow cytometer (Cyflow SL) was modified to allow the detection of light depolarization and it was used for all studies. Other commercially available cytometers (MoFlo, Accuri C6, Attune) were also easily adapted to detect light depolarization, showing that the measurement of this additional parameter can be accomplished in different instruments. In vitro cultures of P. falciparum were established and allowed to further investigate the potential of this novel method. Ring-stage synchronized cultures of P. falciparum were incubated with several antimalarial drugs (chloroquine, mefloquine, quinine, artemisinin, artesunate and pyrimethamine). Analysis of depolarizing events, corresponding to parasitized erythrocytes containing hemozoin, allowed the detection of parasite maturation. Furthermore, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 - 24 hours of incubation. The 50% inhibitory concentrations (IC50) obtained at 24 hours of incubation were comparable to previously reported values. However, these values were most of the times higher than the ones obtained with the already validated HRP2 ELISA assay. Indeed, IC50 values may differ considerably between assays. Different assays measure different parameters to assess parasite growth, at different time-points. Moreover, variations in parasite density and hematocrit as well as the stage-dependent action of antimalarial drugs, may influence these values. Altogether, explaining the differences in IC50 values that are commonly observed. The performance of the hemozoin detection assay in the field was assessed during a 6- month trial performed in Gabon, a malaria endemic country. The trial was conducted in the Centre de Recherches Médicales de Lambaréné – Albert Schweitzer Hospital. On site, an existing flow cytometer (Cyflow SL) was modified to detect light depolarization caused by hemozoin. A total of 46 samples from malaria patients were analyzed during this study. Blood samples were incubated with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing cells was used as maturation indicator and measured at 24, 48 and 72 hours of incubation to determine parasite growth and drug effects. Analysis of ex vivo cultures of parasites obtained from blood samples of malaria patients showed four different growth profiles. The flow cytometric detection of hemozoin allowed to detect drug effects in 39/46 (85%) of samples. In 25 samples drug effects were measurable at 24 hours. In the remaining 14 samples parasite maturation was delayed, and thus drug effects were only detected at 48 hours of incubation. Obtained IC50 values showed that chloroquine-resistant parasites were still common and present in Lambaréné, Gabon but they were fully sensitive to artesunate and artemisinin. Finally, the usefulness of the hemozoin detection assay in the investigation of artemisinin resistance in vitro was also assessed. Artemisinin-resistant (MRA-1240) and sensitive (MRA-1239, 3D7) strains were cultured in vitro. Parasite maturation was determined based on the flow cytometric detection of hemozoin-containing cells. Two different drug assays were performed: 1) standard drug assay: where ring-stage parasites were continuously incubated with increasing concentrations of dihydroartemisinin (DHA) for 48 hours; and 2) pulse assay: where tightly ring-stage synchronized parasites were incubated with a single high-dose (700 nM) of DHA for 6 hours. Results showed that at 24 hours of incubation artemisinin-resistant parasites had increased IC50 values, in comparison to the artemisinin-sensitive strains (15 nM and 8 nM, respectively). Moreover, when parasites were exposed to a high-dose of DHA for 6 hours, increased survival rates associated with artemisinin resistance could be detected after only 30 hours of incubation. Interestingly, it was also observed that artemisinin-resistant parasites do not seem to enter dormancy, as it has been previously suggested by others. Microscopic assessment performed after 72 hours of incubation showed that parasites that survived to a 6-hour exposure to DHA were very close in terms of parasite development to the ones found in the drug free control. Further investigation using more artemisininresistant strains is required to determine whether increased IC50 values correlate with the delayed parasite clearance times observed in the patients; and if the underlying mechanisms of artemisinin resistance is or not related to dormancy. Overall, the work presented in this thesis shows that hemozoin detection by flow cytometry is an alternative, reagent-free and rapid drug assay that overcomes some of the limitations of currently available drug assays for P. falciparum. Moreover, it may also be a useful tool in the study of artemisinin resistance both in culture-adapted strains and, possibly in strains obtained directly from patients. Importantly, this work paves the way for the development and investigation of better tools to assess drug effects and monitor drug resistance in Plasmodium spp. Several novel hemozoin detection platforms are available or under development and should definitely be further explored for their potential to be used as antimalarial drug assays. Furthermore, combination of hemozoin detection with the measurement of other parameters, such as DNA and RNA content and parasite viability may even provide additional important information to reliably determine the developmental stage and metabolic status of parasites and, consequently, detect drug effects. Hopefully, this would lead to the development of an optimal antimalarial drug assay that could play an important role in the fight against malaria

Remote Assessment of the Cardiovascular Function Using Camera-Based Photoplethysmography

Camera-based photoplethysmography (cbPPG) is a novel measurement technique that allows the continuous monitoring of vital signs by using common video cameras. In the last decade, the technology has attracted a lot of attention as it is easy to set up, operates remotely, and offers new diagnostic opportunities. Despite the growing interest, cbPPG is not completely established yet and is still primarily the object of research. There are a variety of reasons for this lack of development including that reliable and autonomous hardware setups are missing, that robust processing algorithms are needed, that application fields are still limited, and that it is not completely understood which physiological factors impact the captured signal. In this thesis, these issues will be addressed. A new and innovative measuring system for cbPPG was developed. In the course of three large studies conducted in clinical and non-clinical environments, the system’s great flexibility, autonomy, user-friendliness, and integrability could be successfully proven. Furthermore, it was investigated what value optical polarization filtration adds to cbPPG. The results show that a perpendicular filter setting can significantly enhance the signal quality. In addition, the performed analyses were used to draw conclusions about the origin of cbPPG signals: Blood volume changes are most likely the defining element for the signal's modulation. Besides the hardware-related topics, the software topic was addressed. A new method for the selection of regions of interest (ROIs) in cbPPG videos was developed. Choosing valid ROIs is one of the most important steps in the processing chain of cbPPG software. The new method has the advantage of being fully automated, more independent, and universally applicable. Moreover, it suppresses ballistocardiographic artifacts by utilizing a level-set-based approach. The suitability of the ROI selection method was demonstrated on a large and challenging data set. In the last part of the work, a potentially new application field for cbPPG was explored. It was investigated how cbPPG can be used to assess autonomic reactions of the nervous system at the cutaneous vasculature. The results show that changes in the vasomotor tone, i.e. vasodilation and vasoconstriction, reflect in the pulsation strength of cbPPG signals. These characteristics also shed more light on the origin problem. Similar to the polarization analyses, they support the classic blood volume theory. In conclusion, this thesis tackles relevant issues regarding the application of cbPPG. The proposed solutions pave the way for cbPPG to become an established and widely accepted technology

Fast widefield techniques for fluorescence and phase endomicroscopy

Thesis (Ph.D.)--Boston UniversityEndomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast widefield imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated

Raman spectroscopy: techniques and applications in the life sciences

Raman spectroscopy is an increasingly popular technique in many areas including biology and medicine. It is based on Raman scattering, a phenomenon in which incident photons lose or gain energy via interactions with vibrating molecules in a sample. These energy shifts can be used to obtain information regarding molecular composition of the sample with very high accuracy. Applications of Raman spectroscopy in the life sciences have included quantification of biomolecules, hyperspectral molecular imaging of cells and tissue, medical diagnosis, and others. This review briefly presents the physical origin of Raman scattering explaining the key classical and quantum mechanical concepts. Variations of the Raman effect will also be considered, including resonance, coherent, and enhanced Raman scattering. We discuss the molecular origins of prominent bands often found in the Raman spectra of biological samples. Finally, we examine several variations of Raman spectroscopy techniques in practice, looking at their applications, strengths, and challenges. This review is intended to be a starting resource for scientists new to Raman spectroscopy, providing theoretical background and practical examples as the foundation for further study and exploration

In vitro modelling of cellular haemozoin and inhibition by β-haematin inhibitors and their derivatives

The discovery of new β-haematin inhibitors has become one focus for researches in response to the resistance of P. falciparum malaria parasites that emerged towards well-known antimalarials. While hundreds of new β-haematin inhibitors have been discovered using detergent mediated high-throughput screening methods, a crucial aspect is understanding exactly how these β-haematin inhibitors behave in the malaria parasite and inhibit the formation of haemozoin. What is known, is that well-known β-haematin inhibitors like chloroquine cause increased amounts of exchangeable haem in the parasite digestive vacuole and form a Fe(III)PPIX-inhibitor complex by accumulating at high concentrations which consequently inhibits parasite growth. Another important focus is on understanding the digestion of haemoglobin and its role in haemozoin formation. This research investigates the in vitro modelling of cellular haemozoin and inhibition by various β-haematin inhibitors across different scaffolds and the role of haemoglobin degradation in P. falciparum malaria parasites. The investigated β-haematin inhibitors resulted in micromolar IC50 (NF54) values and decreased parasite growth with increases in concentration. Using a pyridine-based parasite haem fractionation plate method, these β-haematin inhibitors were shown to target haemozoin formation by causing increased amounts of exchangeable haem that corresponded to decreasing amounts of haemozoin in chloroquine-sensitive parasites. The amounts of exchangeable haem were shown to be inversely proportional to the percentage of parasite growth in the presence of these β-haematin inhibitors. It was apparent that there was a tendency for parasite growth inhibition activity to decrease as the amount of exchangeable haem present in chloroquine sensitive parasites increased, although, the trend was not statistically significant. Moreover, it was observed that experimental cellular accumulation ratio values were low in comparison to chloroquine and amodiaquine. Based on the experimental cellular accumulation ratio values, it was deduced that the accumulation of these β-haematin inhibitors was not primarily due to pH trapping and more complex than previously proposed. Further investigations into the exchangeable haem amounts as a function of intracellular test compound amounts at the IC50 values of these β-haematin inhibitors highlighted that there was an apparent 1:1 relationship with the amount of intracellular exchangeable haem, indicative of complex formation. Transmission electron microscopy images were obtained for untreated parasites that showed intact parasites inside red blood cells with clearly visible haemozoin crystals dispersed throughout the parasite digestive vacuole, whilst, treated parasites showed less defined haemozoin crystals as a result of inhibition. Moreover, electron energy-loss spectroscopy revealed that untreated parasites exhibited a strong iron signal which was associated with haemozoin in the parasite digestive vacuole with a weaker signal attributed to the red blood cell cytoplasm. Similarly, a strong iron signal was shown in the digestive vacuole of treated parasites which was associated with less defined haemozoin crystals. A halo around these haemozoin crystals was observed and was suggested to be indicative of the build-up of exchangeable haem. Additionally, a strong bromine signal attributed to a bromine-containing β-haematin inhibitor, test compound 1, was also observed in the same region as the haemozoin crystals. Overlaid signal distribution maps for iron and bromine showed direct evidence of Fe(III)PPIX and test compound 1, suggesting complexation. High-quality Raman spectra were obtained for the Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetically prepared samples for the Fe(III)PPIX porphyrin dominated spectral region of 1700-500 cm-1 at an excitation wavelength of 532 nm. From the spectra, a putative Fe(III)PPIX-test compound 1 complex was identified and shown to be similar to the synthetically prepared counterpart, haematin-test compound 1 mixture. It was highlighted that a unique peak at 1080 cm-1 indicated π- π interactions between the pyrrole-imidazole ring and thus confirming that the formation of this putative Fe(III)PPIX-inhibitor complex occurs. The confocal Raman true mapping technique proved to be efficient and reliable for imaging the signal distribution of haemozoin at the Raman peak of 754 cm-1 and 1080 cm-1 for the Fe(III)PPIX-test compound 1 complex which co-localized in the digestive vacuole of chloroquine sensitive parasites. Moreover, oxy- and deoxy-haemoglobin was observed to be localized to the red blood cell, where, deoxy-haemoglobin was located on the outer parts of the parasite. Principle component analysis, based on the Raman peak positions, exhibited significant differences in the spectra for Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetic samples where clusters were observed to separate mainly along principle component 1. These data proved that the spectra of the Fe(III)PPIX-test compound 1 complex was the same as its synthetically prepared counterpart but different from the remaining Fe(III)PPIX species. In comparison to the Fe(III)PPIX-test compound 1 complex, the cluster separations were observed to be significant, where, no significant separation was observed for the Fe(III)PPIX-test compound 1 complex and the haematin-test compound 1 mixture. Based on this, it was evident that a Fe(III)PPIX-test compound 1 complex existed in the digestive vacuole of treated chloroquine sensitive parasites. To fully understand the inhibition of haemozoin, the development of a haem pathway model is necessary, but, requires certain prerequisites. Bioinformatics data from PAXdb and ExPASy revealed that chloroquine resistance (Dd2) parasites, containing 1337 previously identified proteins with an average abundance-weighted molecular weight of 40,483 ± 77 g/mol. With this, the protein mass per cell for red blood cells, chloroquine-sensitive and - resistant parasites were consistent across three protein quantification methods was measured and revealed that chloroquine resistant parasites had a significantly higher protein mass per cell than chloroquine sensitive parasites and in turn a higher total number of protein molecules per cell. Aspartic proteases are 4-fold higher in concentration than cysteine proteases with histo-aspartic protease having the highest concentration in chloroquine resistant parasites. Along with these data, a time point quantification for chloroquine sensitive parasites throughout the blood-stage showed that the amount of haemoglobin decreased in a sigmoidal manner and corresponded to a linear increase in the amount of haemozoin and relatively constant exchangeable haem amount. This was consistent with Giemsa smears that showed that for early time points, large initial decreases in the amount of haemoglobin were observed between the early trophozoite to late trophozoite stage

Binding Studies of Near Infrared Cyanine Dyes with Human Serum Albumin and Poly-L-Lysine Using Optical Spectroscopy Methods

The sensitivity of biological studies performed between 190 and 650 nm is greatly reduced due to the autofluorescence of biomolecules and impurities in this region. Therefore, the enhanced signal-to-noise ratios encountered at longer wavelengths makes biological analysis within the near infrared (NIR) region from 650 nm to 1100 nm far more advantageous. This dissertation describes the noncovalent binding interactions of near-infrared (NIR) carbocyanine dyes with human serum albumin (HSA) and poly-L-lysine (PLL) using UV-Vis/NIR absorption spectroscopy, emission spectroscopy, circular dichroism (CD), and fluorescence detected circular dichroism (FDCD). The optical spectroscopy methods used in this work are described in detail in Chapter 1. The various applications of NIR dyes in protein analysis are introduced in Chapter 2. In general, the sensitivity of cyanines to the polarity of their local environment makes them quite suitable for protein labeling schemes. In aqueous media, cyanines have a high propensity for self-association. Yet in the hydrophobic binding sites of globular proteins, these aggregates often dissipate. Absorption and emission spectroscopy can be utilized to observe the differential spectral properties of monomer, intra-molecular and intermolecular aggregates. In Chapter 3, the photophysical properties of bis(cyanine) NIR dyes containing di-, tri-, and tetraethylene glycol linkers were each examined in the presence of HSA are discussed. Variations in chain length as well as probe flexibility were demonstrated through distinct differences in absorption and emission spectra. The observed changes in the spectral properties of the NIR dyes in the presence and absence of HSA were correlated to the physical parameters of the probes\u27 local environment (i.e., protein binding sites and self-association). All three bis-cyanines examined exhibited enhanced fluorescence in the presence of HSA. The bis-cyanine dye containing the tri(ethylene glycol) spacer allowed for a complete overlap of the benzene rings, to form π-π interactions which were observed as intra-molecular H-aggregate bands. The dye exhibited no fluorescence in buffer, owing to the H-aggregation observed in the absorption data. In the presence of HSA, the intra-molecular dimers were disrupted and fluorescence was then detected. The cut-on fluorescence displayed by the dye in the presence of HSA made it ideal for noncovalent labeling applications. The utility of several NIR dyes for use as secondary structural probes was investigated in Chapter 4. NIR dyes were screened thoroughly using UV-Vis/NIR absorption spectroscopy dyes with spectral properties which were sensitive to protein secondary structure models of such as PLL in basic solution. Two NIR dyes were found to be quite sensitive to the structural features of uncharged α- and β-PLL. The chiral discrimination of these probes for basic protein secondary structures was also evaluated through CD measurements within the NIR probes\u27 absorption bands

Hemoglobin contrast in magneto-motive optical doppler tomography, optical coherence tomography, and ultrasound imaging methods and apparatus

Provided herein are systems, methods, and compositions for the use of optical coherence tomography for detection of cells.Board of Regents, University of Texas Syste