Atlikta klinikinės medžiagos spektroskopinė analizė indukuojant autofluorescenciją lazerine spinduliuote. Eksperimento metu naudotas nešiojamas trečios harmonikos generatorius (355nm.). Tiriamoji medžiaga atviros stuburo išvaržos pašalinimo operacijos metu buvo imta iš skirtingų vietų: D- iš disko gilumos, P – išvarža iš po raiščio ir S – laisvas sekvestras. Viso buvo surinkta 29 bandiniai: 9 - D, 12 – P ir 8 S. Eksperimentų metų gauti rezultatai buo lyginami su histologinėmis tos pašios medžiagos išvadomis. Mūsų atliktų tyrymų rezultatai rodo, kad sukeltoji autofluorescencija rodo skirtingas medžiagas sudarančias tarpslankstelinį diską. Manome, kad pritaikius spektroskopinę analizę galima nustatyti disko biocheminės struktūros kitimus, taipogi, netiesiogiai spręsti apie disko degeneracijos procesą. Iš spektro sudedamųjų komponentų galima prelimenariai nustatyti tarpslanksteliio disko išvaržos sausgyslinio žiedo (P grupė) degeneracijos lygmenį, kuris neatsispindi kitais, neinvaziniais atvaizdavimo metodais. Pašalintų tarpslankstelinio disko mėginių spektrinis tyrimas sudaro pagrindą tolimesniems degeneruojančio disko biocheminiams ir spektriniams tyrimams. / The analysis of material of clinical state was made inducing the autofluorescence using the third harmonic 355 nm. laser radiation generator. Experimental material was taken during open vertebral hernia removal procedure. Accordingly to intraoperative findings, all the samples were divided into three groups: D group- disc specimen gathered from inside the disc space; P group- herniation removed from subligamentous space; S group- free sequester in direct contact with epidural vessels. For the final assessment 29 disc specimens were collected. Stained disc sections were evaluated also histologicaly. The results indicate that auto fluorescence refers to different substances constituting intervertebral disc. Spectral analysis can be used to determine biochemical changes content of intervertebral disc matrix, and signs of disc degeneration can be identified indirectly. According to the spectral gaussian components in spectra, degeneration in intervertebral disc (P group) may be determined indirectly. Moreover, spectroscopy may be a new very sensitive tool in determining intervertebral disc degeneration where other non-invasive methods are helpful. Spectroscopic analysis of intervertebral disc removed during open surgery, creates background for further investigation on degenerating intervertebral disc spectral features and biochemical changes.

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L'objectif de ce projet consiste à identifier des molécules fluorescentes, naturellement présentes dans les cellules, et dont l'évolution de leur concentration sera représentative du comportement métabolique de cellules en suspension. Ces molécules pourront servir au suivi du métabolisme cellulaire en temps réel durant une culture. Un besoin pour ce type d'outils se fait sentir afin de pouvoir développer et optimiser des bioprocédés, dont ceux utilisant des cellules végétales indifférenciées en suspension. La première étape fut la sélection et la caractérisation expérimentale de 11 molécules candidates à l'aide d'un spectrophomètre équipé d'un monochromateur quadruple. Après la réalisation des empreintes optiques des molécules, 16 zones d'intérêt dans le spectre d'autofluorescence furent identifiées et suivies dans le temps. Les signaux de la suspension complète, du milieu et des cellules filtrées furent analysés afin d'identifier 10 marqueurs métaboliques, caractérisés par 10 couples de longueurs d'ondes corrélés à des variables de culture. Cette méthode a permis d'identifier dix marqueurs métaboliques dans la suspension complète et laisse entrevoir la possibilité de plusieurs autres dans le milieu et les cellules filtrées. Le fait que certains marqueurs puissent se valider entre eux rajoute à la puissance de cette méthode. Il a donc été démontré que l'autofluorescence des cellules de plante indifférenciées en suspension contient assez d'information pour permettre le suivi de la croissance cellulaire en temps réel. La prochaine étape sera donc de valider ces marqueurs sur d'autres types de cellules.

Microglia are a unique type of immune cell found within the brain, spinal cord and retina. In the healthy brain, their job is to support neurons, defend against infectious microbes, clear extracellular debris and remove dead or dying cells through phagocytosis. This diverse array of functions presents the possibility of unique subsets of microglia existing in the healthy brain, yet none have been described thus far. By utilizing cellular autofluorescence as a discriminating characteristic, we identified two novel subsets of microglia present in the healthy brains of mice and non-human primates. Approximately 70% of microglia displayed autofluorescence (AF+) while the remaining 30% did not (AF–). While the proportion of AF+ and AF– microglia remained constant throughout most of adult life, the autofluorescence intensity increased exclusively in the AF+ subset at an almost linear rate with age. This gain in autofluorescence correlated with equivalent increases in the size and complexity of storage bodies, as detected by transmission electron microscopy and increases in LAMP1 levels, a key component of the lysosomal compartment. As the brain ages, lysosomal storage material builds up inside AF+ microglia, further increasing the accumulation of autofluorescence as a result. The analysis of protein content in autofluorescent subsets revealed that AF+ microglia produced more proteins and enzymes involved in the storage and degradation of waste material, as well as more proteins involved in the regulation of mTOR, a key cellular pathway governing nutrient availability and energy production. Interestingly, the disruption of lysosomal function in microglia through genetic mutations accelerated the accumulation of storage material in AF+ cells, which led to impaired microglia physiology and increased cell death, mimicking the effects observed during advanced aging. Increasing evidence suggests that the accumulation of waste materials inside the brain contributes to diseases of aging and these data are suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

Hemodialysis (HD) is the most frequently used treatment for end-stage renal disease. Despite all efforts to improve the outcomes, the mortality of patients on HD is still high, and this especially is related to cardiovascular diseases (CVD). Glucose degradation products accumulate in plasma and tissue as a result of oxidative stress in these patients. Such accumulation is strongly related to the risk of developing CVD. Tissue deposits of advanced glycation end products (AGE) can be easily assessed by a skin autofluorescence (SAF) technique. SAF is one of the strongest prognostic markers of mortality in HD patients. The aim of this thesis is to examine whether intervention on HD treatment can reduce the load of AGE of these patients. The aim of the first study was to investigate whether changes in SAF appear after a single HD session and if they might be related to changes in plasma AF. Skin and plasma AF (PAF) were measured before and after HD in 35 patients on maintenance HD therapy. Median dialysis time was 4 h (range 3-5.5). SAF was measured noninvasively with an AGE Reader, and plasma AF was measured before and after HD. The HD patients had on average a 65% higher SAF value than age-matched healthy persons (P < 0.001). PAF was reduced by 14% (P < 0.001), whereas SAF was not changed after a single HD treatment. No significant influence of the reduced PAF on SAF levels was found. This suggests that the measurement of SAF can be performed during the whole dialysis period and is not directly influenced by the changes in plasma AF during HD. In study 2 different dialysis filters were compared to clarify whether using a high-flux (HF) dialyzer favors plasma or SAF removal compared to low-flux (LF) dialyzer. Twenty-eight patients were treated with either an HF-HD or LF-HD but otherwise unchanged conditions in a cross-over design. SAF was measured non-invasively with an AGE reader before and after HD. PAF was determined as total and non-protein-bound fractions. Corrections for hemoconcentrations by volume changes were made using the change in serum albumin. Paired and non-paired statistical analyses were used. The different treatments did not change SAF after LF- and HF-dialysis. Total, free, and protein-bound PAF were reduced after a single LF-HD by 21%, 28%, and 17%, respectively (P<.001). After HF-HD total and free PAF was reduced by 5% and 15%, respectively (P<.001), while protein-bound values were unchanged. The LF-HD resulted in a more pronounced reduction of PAF than did HF-HD (P<.001). Serum albumin correlated inversely with PAF in HF-HD. There was no significant change in SAF after dialysis, either with LF or with HF dialysis. Although only limited reductions in PAF were observed, these were more pronounced when performing LF dialysis. These data are not in overwhelming support of the use of HF dialysis in the setting used in this study. In the third study the effect on SAF was investigated using either glucose-containing or glucose-free dialysate. SAF and PAF were measured in patients on HD during standard treatment with a glucose-containing dialysate (n=24). After that, the patients were switched to a glucose-free dialysate for a 2 week period, and new measurements were performed on PAF and SAF. There was an increase of pre-dialysis SAF measured at the beginning of the study compared with the values one month later (as in study 4). By comparing pre- and post-dialysis values there was a significant decrease of SAF only when using glucose-free dialysate. Free PAF decreased independently whether glucose-containing or glucose-free dialysate was used. The important finding was that increase in SAF seemed possible to slow down using glucose-free dialysate. Study 4 was performed to investigate whether there are seasonal variations in SAF on a HD population. SAF was measured non-invasively with an AGE Reader in patients on HD at different seasonal periods during one year such as February-May (N=31), May–August (N=28), August–March (N=25). SAF was measured before HD. Paired statistical analyses were performed between each two periods.  Unexpectedly there was at a median 6% increase in SAF during the winter (p=0.004) and a 11% decrease from 4.0 to 3.5 arbitrary units of the SAF during the summer (p<0.001). The study concluded that SAF shows seasonal variation. The cause of these changes could not be clarified. A beneficial effect may be due to extended exposure to sunlight during the summer and/or to different dietary intakes during the seasons. In conclusion, these interventional studies confirmed that PAF is lowered by dialysis. SAF was only decreased by HD when using glucose-free dialysate. SAF was not influenced by a single HD, with glucose-containing dialysate, independent of using HF or LF filters. These data favor glucose-free dialysate as a possible measure to slow down the progress of tissue AGE compared to glucose-containing dialysate. Longitudinal studies will help to clarify this issue further.

Le premier traitement standard pour les tumeurs cérébrales est la résection chirurgicale. Dans cette procédure un enjeu important demeure, l'identification des berges tumorales pour assurer une résection totale et éviter le risque de récidive pour le patient. A ce jour aucune technique d'imagerie peropératoire est capable de résoudre l'infiltration tumorale du tissu sain. La norme pour le diagnostic des berges tumorales est l'analyse histologique des biopsies. Une méthode ex vivo qui requiert un à plusieurs jours pour fournir ler apport pathologique final, un lapse de temps qui peut s'avérer fatal pour le patient. La microscopie optique a récemment été développer vers une utilisation clinique peropératoire pour répondre à cet enjeu. Dans travail, la technique de microscopie à deux-photons a été préférée pouressayer de répondre à cette problématique. Cette méthode donne accès à deux contrastes d'imagerie, la génération de seconde harmonique et l’émission de fluorescence, qui peuvent être combinés à des mesures quantitatives, tel que la spectroscopie et le temps de vie de fluorescence. Combiner ces quatre modalités de détection donnera une information complète sur la structure et le métabolisme de la région observée. Pour soutenir le développement technique vers une sonde endomicroscopique visant une utilisation peropératoire, les données en résultants doivent être fiables, et se montrer d'un intérêt pour le chirurgien. Par conséquent, une base de données sur le signal d'autofluorescence des tissus a été construite et présentée dans ce manuscrit, avec des algorithmes capables de discriminer de façon fiable les régions tumorales des régions saines. Des algorithmes qui ont montré le potentiel d'être automatisé dans une configuration clinique, afin de fournir une réponse en temps-réel au chirurgien. / The first standard approach for brain tumor treatment is the surgical resection. In this protocol an important challenge remains, the identification of tumor margins to ensure a complete resection and avoid risk of tumor recurrence. Nowadays no intra-operative means of contrast are able to resolve infiltrated regions from healthy tissue. The standard for tumor margin diagnosis is the histological analysis of biopsies. An ex vivo method that requires one to several days to issue a final pathological reports, a time lapse that could be fatal to the patient. Optical microscopy have recently been developed towards an intra-operative clinical use to answer this challenge. In this work, the technique of two-photon microscopy based on the autofluorescence of tissue have been favored. This technique gives access to two imaging contrasts, the second-harmonic generation and emission of fluorescence, and can be combined to quantitative measurements, such as spectroscopy and fluorescence lifetime. The combination of these four modalities of detection will give a complete structural and metabolic information on the observed region. To support the technical development towards an endomicroscopic probe, the resulted data have to be reliable and proved to be of interest for the surgeon. Consequently, an extensive database of the autofluorescence response of brain tumor tissue have been constructed and presented in this manuscript, with algorithms able to discriminate with reliability tumoral from healthy regions. Algorithms that have shown potential to be automatized in a clinical setting, in order to give a real-time answer to the surgeons.

Fluorescence from endogenous molecules and exogenous contrast agents can provide morphological, spectral, and lifetime contrast that indicates disease state in epithelial tissues. Recently, nonlinear microscopy has emerged as a potential tool for the early detection, case-finding, and monitoring of epithelial cancers because it permits non-invasive, three-dimensional fluorescence imaging of subcellular features hundreds of microns deep. This dissertation explores the use of nonlinear microscopy for cancer diagnostics on two fronts: (1) we examine the fundamental limitations governing the maximum nonlinear imaging depth in epithelial tissues, and (2) we investigate the use of a new class of nonlinear contrast agent---plasmonic gold nanoparticles---for molecularly specific imaging of cancer cells. We built and optimized a nonlinear microscope for deep tissue imaging, and studied the image contrast as a function of imaging depth in ex-vivo human biopsies and tissue phantoms. With this system we demonstrated imaging down to 370 [mu]m deep in a human biopsy, which is significantly deeper than imaging depths achieved in comparable studies. We found that the large scattering coefficient and homogenous fluorophore distribution typical of epithelial tissues limit the maximum imaging depth to 3-5 mean free scattering lengths deep in conventional nonlinear microscopy. Beyond this imaging depth, the increasing contribution of out-of-focus emission limits the contrast to insufficient levels for diagnostic imaging. We support these observations with time-dependent Monte Carlo simulations. We exploited the intense interaction of gold nanoparticles with light, enhanced by surface plasmon resonance effects, to create extremely bright nonlinear contrast agents. These contrast agents proved to be several orders of magnitude brighter than the brightest organic fluorophores and at least one order of magnitude brighter than quantum dots. We targeted gold nanoparticles to a biomarker for carcinogenesis and demonstrated molecularly specific imaging of cancer cells. We demonstrated that unlike emission from traditional bandgap fluorophores, nonlinear luminescence from gold nanoparticles was weakly dependent on excitation pulse length for short pulse durations. This finding supports the hypothesis that nonlinear excitation in plasmonic nanoparticles involves sequential rather than simultaneous absorption of excitation photons. The remarkable brightness of gold nanoparticles makes them an attractive contrast agent for nonlinear diagnostics. / text

Cancer is the second leading cause of death in the United States, and its early diagnosis is critical to improving treatment options and patient outcomes. In autofluorescence (AF) imaging, light of controlled wavelengths is projected onto tissue, absorbed by specific molecules, and re-emitted at longer wavelengths. Images of re-emitted light are used together with spectral information to infer tissue functional information and diagnosis. This dissertation describes AF imaging studies of three different organs using data collected from fresh human surgical specimens. In the ovary study, illumination was at 365 nm, and images were captured at 8 emission wavelengths. Measurements from a multispectral imaging system and fiber optic probe were used to map tissue diagnosis at every image pixel. For the colon and pancreas studies, instrumentation was developed extending AF imaging capability to sub-300 nm excitation. Images excited in the deep UV revealed tryptophan and protein content which are believed to change with disease state. Several excitation wavelength bands from 280 nm to 440 nm were investigated. Microscopic AF images collected in the pancreas study included both cultured and primary cells. Several findings are reported. A method of transforming fiber optic probe spectra for direct comparison with imager spectra was devised. Normalization of AF data by green reflectance data was found useful in correcting hemoglobin absorption. Ratio images, both AF and reflectance, were formulated to highlight growths in the colon. Novel tryptophan AF images were found less useful for colon diagnostics than the new ratio techniques. Microscopic tryptophan AF images produce useful visualization of cellular protein content, but their diagnostic value requires further study.

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