While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone retinae. Previous work from the Chang lab identified three visual pigments expressed in the photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T. proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein expression. T. proximus have four types of morphological cones: double cones, large single cones, small single cones, and very small single cones. Some small single cones have rod-like features, such as rod-like outer-segment membranes and a lack of micro-droplets. Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with λmax at 485nm, which corresponds to microspectrophotometry measurement from some small single cones. Current results suggest that small single cones of T. proximus may have evolved from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

Neste trabalho, foram comparadas as cinéticas de adsorção da enolase (2-fosfo-D-glicerato hidrolase) sobre substratos hidrofílicos (placas de silício não modificadas ou silanizadas com aminopropilsilano (APS)) com aquelas sobre substratos hidrofóbicos (placas de silício silanizadas com trimetilclorosilano (TMCS) ou recobertas com filme de PS (poliestireno)). O efeito da forma do substrato (plano x esférico) sobre a cinética de adsorção também foi estudado. Os substratos esféricos foram esferas de vidro não modificadas (caráter hidrofílico) e silanizadas com TMCS (caráter hidrofóbico). As curvas de cinética de adsorção em substratos planos obtidas por elipsometria in situ mostraram que o processo ocorre em três etapas: (1) difusão das moléculas para a interface sólido/líquido, (2) formação de uma monocamada adsorvida e (3) adsorção de outras moléculas sobre a monocamada e formação de multicamadas. As isotermas mostraram que a enolase não possui adesão preferencial em substratos hidrofílicos ou hidrofóbicos. A etapa (1) pode ser descrita pelo modelo de adsorção seqüencial aleatória, enquanto que as etapas (2) e (3) podem ser descritas pelo modelo de adsorção seqüencial cooperativa. Não foi observada influência da força iônica. Contudo, imagens da topografia das superfícies recobertas por enolase obtidas por microscopia de força atômica (in situ e no ar) mostraram que os agregados de moléculas adsorvidas podem se apresentar na forma esférica (força iônica alta) ou como fibrilas (força iônica baixa). Medidas de espalhamento de raios-X a baixo ângulo (SAXS) de uma solução de enolase (6 g/L NaCl 0,001 mol/L) mostraram que as moléculas possuem raio de giro de 29 Å. Portanto, a agregação é induzida pelas propriedades da superfície da monocamada e pela força iônica do meio. Medidas de ângulo de contato mostraram que substratos inicialmente hidrofóbicos se tornaram hidrofílicos após adsorção da enolase, enquanto que os hidrofílicos apresentaram tendência oposta. Medidas de espectroscopia de fotoelétrons de raios-X evidenciaram que a adsorção sobre silício é mais rápida do que sobre PS, corroborando com os resultados obtidos por elipsometria. A influência do pH na adsorção da enolase em silício e APS mostraram que a adsorção é máxima quando o valor de pH é próximo ao ponto isoelétrico da enzima. A cinética de adsorção da enolase em substratos esféricos hidrofílicos e hidrofóbicos, acompanhada por espectrofotometria UV-vis, mostrou que a quantidade de material adsorvido O nestas superfícies aumenta com o tempo de adsorção e concentração inicial de enolase em solução (efeito de cooperativismo), sendo que o valor final é muito mais elevado nos substratos esféricos do que nos planos. Pela metodologia utilizada não se pôde observar os três estágios característicos da cinética de adsorção obtida para substratos planos. A influência da força iônica somente foi observada na adsorção sobre os substratos esféricos em sistemas concentrados (cenolase > 0,5g/L). As moléculas de enolase permanecem ativas após adsorção nos substratos estudados. / This work aimed to compare the adsorption behavior of enolase (2-phospho-D- glycerate hydrolase) onto hydrophilic (silicon wafers and amino-terminated surfaces (APS)) and hydrophobic planar substrates (polystyrene (PS) film, TMCS). The effect of the substrate shape (planar x spherical) was also studied. The spherical substrates were glass beads, native and modified with TMCS, with hydrophilic and hydrophobic characters, respectively. The adsorption kinetics of enolase onto planar substrates (obtained by means of in situ ellipsometry) presented three distinct regions: (i) a diffusion controlled step, (ii) monolayer formation evidenced by an adsorption plateau and (iii) continuous, irreversible and asymptotic increase of the adsorbed amount with time. The early stages were described by the random sequential adsorption model (RSA), while the cooperative sequential adsorption (CSA) model described regions (ii) and (iii). The adsorption isotherms show that enolase has no preferential adhesion onto hydrophilic or hydrophobic substrates. No significant influence of ionic strength was observed on the adsorption behavior of enolase onto the planar substrates. On the other hand, atomic force microscopy (AFM) showed that at long adsorption time and low ionic strength enolase monolayer induced fibrillation of the incoming molecules. Such effect was not observed at high ionic strength. Increasing the adsorption time, aggregates appeared on the surface, suggesting multilayer formation. Small angle X-ray scattering (SAXS) measurements of enolase (c = 6.0g/L) in NaCl 0.00 1 mol/L solution yielded radius of gyration of 29 Å, confirming that aggregation was probably induced by the surface of enolase monolayer and screening effects. Contact angle measurements showed that PS surfaces became hydrophilic and silicon surfaces turned hydrophobic after the formation of the enolase biofilm. XPS measurements showed that enolase adsorption is faster onto hydrophilic silicon wafers than onto hydrophobic PS fim, corroborating with the ellipsometric measurements. The study of the influence of pH on the enolase adsorption on silicon and APS surfaces showed that was the highest pH was close to the enzyme isoelectric point. The adsorption kinetic curves of enolase onto spherical substrates (obtained by means of UV-vis spectrophotometry) showed that the adsorbed amount (F) increased as function of adsorption time and initial concentration of enolase. The highest F value was obtained on spherical substrates. The three adsorption steps, characteristic of enolase adsorption, could not be observed by means of the methodology used. The influence of ionic strength was observed only in concentrated enolase solutions (cenolase 0.5g/L). The immobilized enolase molecules kept their enzymatic activity, regardless the type of substrate.

Physisorption and reaction at silicon surfaces of a series of brominated organic molecules: bromoethane, 1,2-dibromoethane, 1-bromopropane, 1-bromobutane and 1-bromopentane were examined by Scanning Tunneling Microscopy (STM). On Si(111)-7×7, a widely-spaced “one-per-corner-hole” pattern was observed, formed by the physisorption and reaction of several alkyl bromides. This “one-per-corner-hole” pattern suggested long-range repulsion between the adsorbates. Density Functional Theory (DFT) calculations, performed by others in parallel with these experiments, showed that this long-range repulsion was due to lateral charge transfer in the Si(111)-7×7 surface consequent on the physisorption of an alkyl bromide or chemisorption of a Br atom. The reaction rate of bromine ‘abstraction’ (transfer of a Br-atom from the adsorbate to the silicon) was examined for two physisorbed states of 1 bromopentane on Si(111)-7×7, one vertical and one horizontal, each distinguishable by STM. The energy barrier was found to be significantly lower for abstraction of Br-atom from the vertical than for the horizontal 1 bromopentane, both for thermal and electron-induced reaction. This finding accords with previous DFT calculations for methyl bromide, for which theory exhibited a clear preference for a vertical transition state in the bromination of Si(111)-7×7. The effect of alkyl chain-length on the rate of thermally-induced dissociative attachment reactions was investigated for a series of primary bromo-alkanes (bromoethane, 1-bromopropane and 1-bromobutane) on a different face of silicon; Si(100)-c(4×2). These three bromo-alkanes all physisorbed exclusively ‘inter-row’, bridging the gap between Si dimer-rows of Si(100)-c(4×2). Thermal reaction was highly ‘localized’, i.e. the chemisorbed Br-atom was formed directly below the parent bromo-alkane. The thermal barrier heights were found experimentally to increase systematically with chain length. This trend was interpreted, on the basis of DFT calculations performed by the author, as being due to the extra energy required to lift the alkyl group in going from the initial physisorbed state to the more-nearly vertical transition state.

Physisorption and reaction at silicon surfaces of a series of brominated organic molecules: bromoethane, 1,2-dibromoethane, 1-bromopropane, 1-bromobutane and 1-bromopentane were examined by Scanning Tunneling Microscopy (STM). On Si(111)-7×7, a widely-spaced “one-per-corner-hole” pattern was observed, formed by the physisorption and reaction of several alkyl bromides. This “one-per-corner-hole” pattern suggested long-range repulsion between the adsorbates. Density Functional Theory (DFT) calculations, performed by others in parallel with these experiments, showed that this long-range repulsion was due to lateral charge transfer in the Si(111)-7×7 surface consequent on the physisorption of an alkyl bromide or chemisorption of a Br atom. The reaction rate of bromine ‘abstraction’ (transfer of a Br-atom from the adsorbate to the silicon) was examined for two physisorbed states of 1 bromopentane on Si(111)-7×7, one vertical and one horizontal, each distinguishable by STM. The energy barrier was found to be significantly lower for abstraction of Br-atom from the vertical than for the horizontal 1 bromopentane, both for thermal and electron-induced reaction. This finding accords with previous DFT calculations for methyl bromide, for which theory exhibited a clear preference for a vertical transition state in the bromination of Si(111)-7×7. The effect of alkyl chain-length on the rate of thermally-induced dissociative attachment reactions was investigated for a series of primary bromo-alkanes (bromoethane, 1-bromopropane and 1-bromobutane) on a different face of silicon; Si(100)-c(4×2). These three bromo-alkanes all physisorbed exclusively ‘inter-row’, bridging the gap between Si dimer-rows of Si(100)-c(4×2). Thermal reaction was highly ‘localized’, i.e. the chemisorbed Br-atom was formed directly below the parent bromo-alkane. The thermal barrier heights were found experimentally to increase systematically with chain length. This trend was interpreted, on the basis of DFT calculations performed by the author, as being due to the extra energy required to lift the alkyl group in going from the initial physisorbed state to the more-nearly vertical transition state.

While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone retinae. Previous work from the Chang lab identified three visual pigments expressed in the photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T. proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein expression. T. proximus have four types of morphological cones: double cones, large single cones, small single cones, and very small single cones. Some small single cones have rod-like features, such as rod-like outer-segment membranes and a lack of micro-droplets. Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with λmax at 485nm, which corresponds to microspectrophotometry measurement from some small single cones. Current results suggest that small single cones of T. proximus may have evolved from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

Neste trabalho, foram comparadas as cinéticas de adsorção da enolase (2-fosfo-D-glicerato hidrolase) sobre substratos hidrofílicos (placas de silício não modificadas ou silanizadas com aminopropilsilano (APS)) com aquelas sobre substratos hidrofóbicos (placas de silício silanizadas com trimetilclorosilano (TMCS) ou recobertas com filme de PS (poliestireno)). O efeito da forma do substrato (plano x esférico) sobre a cinética de adsorção também foi estudado. Os substratos esféricos foram esferas de vidro não modificadas (caráter hidrofílico) e silanizadas com TMCS (caráter hidrofóbico). As curvas de cinética de adsorção em substratos planos obtidas por elipsometria in situ mostraram que o processo ocorre em três etapas: (1) difusão das moléculas para a interface sólido/líquido, (2) formação de uma monocamada adsorvida e (3) adsorção de outras moléculas sobre a monocamada e formação de multicamadas. As isotermas mostraram que a enolase não possui adesão preferencial em substratos hidrofílicos ou hidrofóbicos. A etapa (1) pode ser descrita pelo modelo de adsorção seqüencial aleatória, enquanto que as etapas (2) e (3) podem ser descritas pelo modelo de adsorção seqüencial cooperativa. Não foi observada influência da força iônica. Contudo, imagens da topografia das superfícies recobertas por enolase obtidas por microscopia de força atômica (in situ e no ar) mostraram que os agregados de moléculas adsorvidas podem se apresentar na forma esférica (força iônica alta) ou como fibrilas (força iônica baixa). Medidas de espalhamento de raios-X a baixo ângulo (SAXS) de uma solução de enolase (6 g/L NaCl 0,001 mol/L) mostraram que as moléculas possuem raio de giro de 29 Å. Portanto, a agregação é induzida pelas propriedades da superfície da monocamada e pela força iônica do meio. Medidas de ângulo de contato mostraram que substratos inicialmente hidrofóbicos se tornaram hidrofílicos após adsorção da enolase, enquanto que os hidrofílicos apresentaram tendência oposta. Medidas de espectroscopia de fotoelétrons de raios-X evidenciaram que a adsorção sobre silício é mais rápida do que sobre PS, corroborando com os resultados obtidos por elipsometria. A influência do pH na adsorção da enolase em silício e APS mostraram que a adsorção é máxima quando o valor de pH é próximo ao ponto isoelétrico da enzima. A cinética de adsorção da enolase em substratos esféricos hidrofílicos e hidrofóbicos, acompanhada por espectrofotometria UV-vis, mostrou que a quantidade de material adsorvido O nestas superfícies aumenta com o tempo de adsorção e concentração inicial de enolase em solução (efeito de cooperativismo), sendo que o valor final é muito mais elevado nos substratos esféricos do que nos planos. Pela metodologia utilizada não se pôde observar os três estágios característicos da cinética de adsorção obtida para substratos planos. A influência da força iônica somente foi observada na adsorção sobre os substratos esféricos em sistemas concentrados (cenolase > 0,5g/L). As moléculas de enolase permanecem ativas após adsorção nos substratos estudados. / This work aimed to compare the adsorption behavior of enolase (2-phospho-D- glycerate hydrolase) onto hydrophilic (silicon wafers and amino-terminated surfaces (APS)) and hydrophobic planar substrates (polystyrene (PS) film, TMCS). The effect of the substrate shape (planar x spherical) was also studied. The spherical substrates were glass beads, native and modified with TMCS, with hydrophilic and hydrophobic characters, respectively. The adsorption kinetics of enolase onto planar substrates (obtained by means of in situ ellipsometry) presented three distinct regions: (i) a diffusion controlled step, (ii) monolayer formation evidenced by an adsorption plateau and (iii) continuous, irreversible and asymptotic increase of the adsorbed amount with time. The early stages were described by the random sequential adsorption model (RSA), while the cooperative sequential adsorption (CSA) model described regions (ii) and (iii). The adsorption isotherms show that enolase has no preferential adhesion onto hydrophilic or hydrophobic substrates. No significant influence of ionic strength was observed on the adsorption behavior of enolase onto the planar substrates. On the other hand, atomic force microscopy (AFM) showed that at long adsorption time and low ionic strength enolase monolayer induced fibrillation of the incoming molecules. Such effect was not observed at high ionic strength. Increasing the adsorption time, aggregates appeared on the surface, suggesting multilayer formation. Small angle X-ray scattering (SAXS) measurements of enolase (c = 6.0g/L) in NaCl 0.00 1 mol/L solution yielded radius of gyration of 29 Å, confirming that aggregation was probably induced by the surface of enolase monolayer and screening effects. Contact angle measurements showed that PS surfaces became hydrophilic and silicon surfaces turned hydrophobic after the formation of the enolase biofilm. XPS measurements showed that enolase adsorption is faster onto hydrophilic silicon wafers than onto hydrophobic PS fim, corroborating with the ellipsometric measurements. The study of the influence of pH on the enolase adsorption on silicon and APS surfaces showed that was the highest pH was close to the enzyme isoelectric point. The adsorption kinetic curves of enolase onto spherical substrates (obtained by means of UV-vis spectrophotometry) showed that the adsorbed amount (F) increased as function of adsorption time and initial concentration of enolase. The highest F value was obtained on spherical substrates. The three adsorption steps, characteristic of enolase adsorption, could not be observed by means of the methodology used. The influence of ionic strength was observed only in concentrated enolase solutions (cenolase 0.5g/L). The immobilized enolase molecules kept their enzymatic activity, regardless the type of substrate.

Volumetric optical microscopy has the advantages of quantitative and global measurement of three-dimensional (3D) biological specimens with high spatial resolution and minimum invasion. However, current volumetric imaging technologies based on light transmission, scattering or fluorescence cannot reveal specimen’s chemical distribution that brings insights to study the chemical events in organisms and their metabolism, functionality, and development. Stimulated Raman scattering (SRS) microscopy allowing visualization of chemical contents based on their intrinsic molecular vibrations is an emerging imaging technology to provide rapid label-free volumetric chemical imaging. This dissertation describes three methodologies for developing advanced volumetric SRS imaging technologies to address the challenges of imaging in vivo samples, imaging speed, and axial resolution. In the first methodology, SRS volumetric imaging is enabled by axially scanning the laser foci for sectioning different depth layers. In Chapter 2, we utilize a piezo objective positioner to drive the objective. Combining with the tissue clearance technique, we realize volumetric SRS imaging up to 500 µm depth in brain tissues showing the potential for 3D staining-free histology. The limitations of piezo scanning are slow speed and disturbance to in vivo samples while rapidly scanning the objective. To tackle the limitations, in Chapter 3, we develop a remote-focusing volumetric SRS microscope based on a deformable mirror and adaptive optics optimization, allowing focal scanning without physically moving the objective or sample. We demonstrate in vivo monitoring of chemical penetration in human sweat pores. In the second methodology, instead of axially scanning the laser foci, the SRS volumetric imaging is enabled by projection imaging with extended depth-of-focus (DOF) beams such as Bessel beams and low numerical-aperture beams. The extended DOF beams integrate SRS signals along the propagation direction to form projection images; thus, a single lateral scan obtains the volumetric chemical information, significantly increasing the volumetric imaging speed for measuring chemical content over a large volume. In Chapter 4, we describe a stimulated Raman projection microscope for fast quantitation of chemicals in a 3D volume. However, projection imaging intrinsically loses axial resolution. We addressed the limitation by developing SRS projection tomography. Mimicking computed tomography, the axial information is reconstructed by angle-dependent projection images obtained by sequentially rotating the sample in a capillary glass tube within the SRS focus. Nevertheless, sample rotation is complicated and not compatible with in vivo samples. To address the difficulty, in Chapter 5, we develop tilted-angle-illuminated stimulated Raman projection tomography which utilizes tilted-angle beams with a tilted angle respected to the optical axis of the objective to obtain angle-dependent projections. This scheme is free of sample rotation and enables fast projection scanning for pushing the imaging speed. The calibration approach and vector-field back-projection algorithm are developed for the multi-view tomographic reconstruction. In the third methodology, we improve the spatial resolution in miniature volumetric SRS imaging via the innovation of metasurface photonics. In developing an SRS endoscope for volumetric chemical imaging inside the human body, the axial resolution deteriorates due to chromatic and monochromatic aberrations induced by poorly made miniature objective lenses. In Chapter 6, we develop a silicon metasurface tailored for compensating the phase errors between the pump and Stokes wavelengths of a singlet refractive lens. Integrating the metasurface with the refractive lens, the hybrid achromatic metalens is compact and provides nearly diffraction-limit resolution, demonstrating a way for developing high resolution chemical imaging endoscopy.