To reduce the image blurring which originates from contributions of a cylindrical array of photolysis events in a photo-induced experiment, a variant of fluorescence imaging techniques has been developed to study photodissociation dynamics and collisional relaxation processes in the bulk. The experimental arrangement utilizes sliced imaging techniques of photofragments by the laser-induced fluorescence detection scheme. An unconventional procedure is employed to guide the photolysis laser in the viewing direction of the imaging detector with a proper obstruction. The sliced image in the direction perpendicular to the photolysis laser is equivalent to a two-dimensional projection of the fluorescence image of photoproducts from a single photolysis center. Experimental images of state-selected CN photofragments from the ICN photodissociation are presented to illustrate the versatility of the present method.

DNA-modified surfaces have been widely studied for microarray and biosensor applications, in particular sequence-specific detection of DNA, for which electrochemical and optical signs can be produced. Variations in the organization and surface density of adsorbed DNA are known to affect the sensitivity and reliability of assays performed using such surfaces, however most measurements of such surfaces to date have little to no spatial resolution, limiting the information that can be gathered regarding the heterogeneity of the organization of adsorbed DNA molecules. We have applied in situ epi-fluorescence microscopic imaging in conjunction with electrochemical measurements to fluorescently labelled thiolate DNA, adsorbed on polycrystalline gold electrodes with a mercaptohexanol (MCH) passive layer. Spatially resolved information on the organization of adsorbed DNA on the surface is gathered within an area measuring 520by 730micrometres with a 0.96 micrometre resolution. The technique has enabled us to investigate "hotspots" (regions of anomalously bright fluorescence) and regional variation in fluorescence; since molecular fluorescence is quenched as a function of distance from the metal substrate, potential modulation with consequent DNA reorientation or layer specificity of the adsorption. Furthermore, an alternative means to the conventional preparation of thiolate-DNA / MCH monolayers has been developed. In this new method, a gold substrate passivated with MCH is subsequently immersed in an aqueous solution of 5'hexylthiol modified DNA. Through a ligand exchange process, DNA is immobilized forming a mixed MCH / DNA monolayer. Samples prepared via the new method display fewer hotspots and improved fluorescence switching of the DNA during electromodulation for samples made with single stranded (ss) DNA and with double stranded (ds) DNA. Measurement of the DNA surface concentration using ruthenium (III) hexaammine chloride with cyclic voltammetry for self assembled monolayers (SAMs) prepared via the new method are on the order of 1% of the maximum grafting density obtainable for both ssDNA and dsDNA by conventional methods.

DNA-modified surfaces have been widely studied for microarray and biosensor applications, in particular sequence-specific detection of DNA, for which electrochemical and optical signs can be produced. Variations in the organization and surface density of adsorbed DNA are known to affect the sensitivity and reliability of assays performed using such surfaces, however most measurements of such surfaces to date have little to no spatial resolution, limiting the information that can be gathered regarding the heterogeneity of the organization of adsorbed DNA molecules. We have applied in situ epi-fluorescence microscopic imaging in conjunction with electrochemical measurements to fluorescently labelled thiolate DNA, adsorbed on polycrystalline gold electrodes with a mercaptohexanol (MCH) passive layer. Spatially resolved information on the organization of adsorbed DNA on the surface is gathered within an area measuring 520by 730micrometres with a 0.96 micrometre resolution. The technique has enabled us to investigate "hotspots" (regions of anomalously bright fluorescence) and regional variation in fluorescence; since molecular fluorescence is quenched as a function of distance from the metal substrate, potential modulation with consequent DNA reorientation or layer specificity of the adsorption. Furthermore, an alternative means to the conventional preparation of thiolate-DNA / MCH monolayers has been developed. In this new method, a gold substrate passivated with MCH is subsequently immersed in an aqueous solution of 5'hexylthiol modified DNA. Through a ligand exchange process, DNA is immobilized forming a mixed MCH / DNA monolayer. Samples prepared via the new method display fewer hotspots and improved fluorescence switching of the DNA during electromodulation for samples made with single stranded (ss) DNA and with double stranded (ds) DNA. Measurement of the DNA surface concentration using ruthenium (III) hexaammine chloride with cyclic voltammetry for self assembled monolayers (SAMs) prepared via the new method are on the order of 1% of the maximum grafting density obtainable for both ssDNA and dsDNA by conventional methods.

DNA-modified surfaces have been widely studied for microarray and biosensor applications, in particular sequence-specific detection of DNA, for which electrochemical and optical signs can be produced. Variations in the organization and surface density of adsorbed DNA are known to affect the sensitivity and reliability of assays performed using such surfaces, however most measurements of such surfaces to date have little to no spatial resolution, limiting the information that can be gathered regarding the heterogeneity of the organization of adsorbed DNA molecules. We have applied in situ epi-fluorescence microscopic imaging in conjunction with electrochemical measurements to fluorescently labelled thiolate DNA, adsorbed on polycrystalline gold electrodes with a mercaptohexanol (MCH) passive layer. Spatially resolved information on the organization of adsorbed DNA on the surface is gathered within an area measuring 520by 730micrometres with a 0.96 micrometre resolution. The technique has enabled us to investigate "hotspots" (regions of anomalously bright fluorescence) and regional variation in fluorescence; since molecular fluorescence is quenched as a function of distance from the metal substrate, potential modulation with consequent DNA reorientation or layer specificity of the adsorption. Furthermore, an alternative means to the conventional preparation of thiolate-DNA / MCH monolayers has been developed. In this new method, a gold substrate passivated with MCH is subsequently immersed in an aqueous solution of 5'hexylthiol modified DNA. Through a ligand exchange process, DNA is immobilized forming a mixed MCH / DNA monolayer. Samples prepared via the new method display fewer hotspots and improved fluorescence switching of the DNA during electromodulation for samples made with single stranded (ss) DNA and with double stranded (ds) DNA. Measurement of the DNA surface concentration using ruthenium (III) hexaammine chloride with cyclic voltammetry for self assembled monolayers (SAMs) prepared via the new method are on the order of 1% of the maximum grafting density obtainable for both ssDNA and dsDNA by conventional methods. / Science, Faculty of / Chemistry, Department of / Graduate

In this Medical Physics M.Sc. project, a near-infrared (NIR) fluorescence imaging system was built for in-vivo diagnosis and evaluation of pigmented skin abnormalities and diseases. Light coming from a 785 nm diode laser is coupled into a ring light guide to uniformly illuminate the skin surface with a field-of-view (FOV) of 25 mm diameter. The diffuse reflectance and emitted fluorescence photons are collected by an NIR-sensitive CCD camera, with computer-controlled filter switch to select between reflectance mode and fluorescence mode. Both reflectance and fluorescence images of skin disorders were obtained with an exposure time of 2 seconds. The results show that cutaneous melanin in pigmented skin disorders emits higher NIR autofluorescence (AF) than surrounding normal tissue. This finding challanged the conventional concept that melanin is a non-fluorescence substance. The developed NIR autofluorescence imaging method also provided a new and direct way to characterize cutaneous melanin and can potentially be used for evaluation and diagnosis of pigmented skin diseases and skin cancers, such as melanoma.

In this Medical Physics M.Sc. project, a near-infrared (NIR) fluorescence imaging system was built for in-vivo diagnosis and evaluation of pigmented skin abnormalities and diseases. Light coming from a 785 nm diode laser is coupled into a ring light guide to uniformly illuminate the skin surface with a field-of-view (FOV) of 25 mm diameter. The diffuse reflectance and emitted fluorescence photons are collected by an NIR-sensitive CCD camera, with computer-controlled filter switch to select between reflectance mode and fluorescence mode. Both reflectance and fluorescence images of skin disorders were obtained with an exposure time of 2 seconds. The results show that cutaneous melanin in pigmented skin disorders emits higher NIR autofluorescence (AF) than surrounding normal tissue. This finding challanged the conventional concept that melanin is a non-fluorescence substance. The developed NIR autofluorescence imaging method also provided a new and direct way to characterize cutaneous melanin and can potentially be used for evaluation and diagnosis of pigmented skin diseases and skin cancers, such as melanoma.

To date, surgery is the most common and successful way to treat cancer. Tumour identification during surgery, however, can be challenging as it relies on the surgeon’s ability to differentiate healthy from diseased tissue, based on visual appearance and palpation. Additional contrast mechanisms are needed to further improve cancer detection during surgery. In this work, I explore the possibility of improving surgical outcomes by using intra-operative fluorescence imaging technologies to identify otherwise invisible lesions. A theoretical model is developed to quantify and characterise the imaging performance of fluorescence image-guided surgery (FIGS) and to guide the development of imaging systems. This model shows excellent potential for performance characterisation of FIGS devices, particularly when small lesions are involved. The design, development and testing of FIGS devices for open and keyhole surgery are described. These devices exploit near infrared (NIR) wavelengths to achieve a superior depth penetration while minimising tissue autofluorescence. Unlike existing systems, the devices described in this work use a single miniaturised camera to simultaneously detect bright-field and fluorescence from multiple dyes. Practical tests indicate nano-molar detection of clinically approved fluorescence dyes. The proposed technology is tested in a clinical study for detecting the sentinel lymph node (SLN) in gynaecological cancers. Results using two non-specific NIR dyes show excellent SLN detection rate in real time during open surgery and laparoscopy. In addition, multi-spectral fluorescence allows independent visualisation of different lymphatic pathways, crucial for understanding the mechanisms of metastasis through the lymphatic system. The FIGS devices are also used to test novel tumour-specific markers in vivo, ex vivo and in vitro. Promising results are reported, suggesting that this imaging technology is suitable for fluorescence molecular imaging. In conclusion, I report the development and applications of a novel multi-spectral FIGS technology that can effectively improve outcomes in surgical oncology.

The photodissociation dynamics of cyanogen iodide (ICN) in the A continuum was studied by the three-dimensional sliced fluorescence imaging (3DSFI) method. The fourth harmonic of a Nd:YAG laser (266 nm) was used to dissociate a low pressure (5 mTorr) sample of ICN, and the resulting CN photofragments were probed by optical-optical double resonance (OODR) detection scheme. A theoretical framework on the emitted fluorescence intensity has been developed to analyze the experimentally obtained image. The vector correlations among the ICN parent molecule transition dipole moment £g, the CN fragment recoil velocity v and the CN rotational angular moment J can be revealed.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23215号 / 工博第4859号 / 新制||工||1758(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 大江 浩一, 教授 近藤 輝幸, 教授 深澤 愛子 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21799号 / 工博第4616号 / 新制||工||1719(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 跡見 晴幸, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM