The juxtaglomerular niche occupied by renin cells (RCN) plays an important role in glomerular repair but the precise temporal and spatial interrelations remain unclear. This study proposes the hypothesis of a local intra-extraglomerular regenerative feedback system and establishes a new quantifiable system for RCN responses in individual glomeruli in vivo. A strictly intraglomerular twophoton laser-induced injury model was established. Labeled renin cells (RC) in transgenic renin reporter mice were fate-traced in healthy and injured glomeruli over several days by intravital microscopy and quantified via new three-dimensional image processing algorithms based on ray tracing. RC in healthy glomeruli demonstrated dynamic extraglomerular protrusions. Upon intraglomerular injury the corresponding RCN first increased in volume and then increased in area of dynamic migration up to threefold compared to their RCN. RC started migration reaching the site of injury within 3 hours and acquired a mesangial cell phenotype without losing physical RCN-contact. During intraglomerular repair only the corresponding RCN responded via stimulated neogenesis, a process of de novo differentiation of RC to replenish the RCN. Repeated continuous intravital microscopy provides a state-of-the-art tool to prove and further study the local intraglomerular RCN repair feedback system in individual glomeruli in vivo in a quantifiable manner.

[pt] O bunching de fótons é um dos mais celebrados efeitos de interferência de dois fóotons, associado à tendéncia de fótons indistinguíveis de tomarem o mesmo caminho quando há uma superposição dos pacotes de onda em um combinador de feixes óptico simétrico. Nós exploramos o fenômeno de interferência de dois fótons e mostramos que: a característica espectral de uma fonte de luz pode ser determinar através da técnica de Espectroscopia de Transformada de Fourier de Poucos Fótons de alta resolução que se mostra como uma técnica útil para a caracterização espectral de fontes ópticas débeis abaixo do limite coberto por técnicas clássicas de batimento heteródino; uma fonte de fótons com estatística sub-Poisson, a Fonte de Fótons Anunciadas com Óptica Linear, pode ser construída baseada apenas em óptica linear e estados coerentes atenuados uma vez que os anúncios sejam sintonizados nos picos de coincidência de um interferograma de Hong-Ou-Mandei quando o interferômetro é alimentado com estados em frequências diferentes; uma modificação do interferômetro de Hong-Ou-Mandel produz um aumento no número de coincidências, ao invés de sua diminuição, quando os pacotes de onda estão perfeitamente superpostos no interferômetro e o anúncio de fótons sintonizados nesse pico gera um feixe com distribuição sub-Poisson. A descrição matemática de cada experimento é detalhada e uma revisão extensa das ferramentas teóricas e práticas necessárias para o entendimento dos resultados é apresentada. / [en] Photon bunching is one of the most celebrated effects of two-photon interference, related to the tendency of indistinguishable photons to take the same path when there is a wave-packet overlapping in a symmetric beam splitter. Photon antibunching, the counterpart of photon bunching is, on turn, a desired effect in many applications such as single-photon generation. We explore the two-photon interference phenomena and show that: the spectral characteristics of a light source can be determined with a high resolution Few-Photon Fourier Transform Spectroscopy which proves to be a useful asset for spectral characterization of faint optical sources below the range covered by classical heterodyne beating techniques; a sub-Poisson photon source, the Linear-Optic Heralded Photon Source, can be constructed based only on linear optics and weak coherent states by time-tuning a Hong-Ou-Mandel interferometer fed with frequency-displaced coherent states and yields a second-order correlation function at zero time below one; a modified version a the Hong-Ou-Mandel interferometer produces a peak of coincidences instead of dip when the wave-packets are perfectly overlapped and the announcement of photons time-tuned to this coincidence peak yield an antibunched photon stream. The mathematical description of each experiment is detailed and an extensive review of the most important theoretical and practical tools for understanding of the results is presented.

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[ES] La presente tesis doctoral titulada "Nuevas sondas, portadores y pro-fármacos dirigidos a células senescentes in vivo" se centra en el diseño, preparación, caracterización y evaluación de sondas basadas en fluoróforos funcionalizados o en nanopartículas, así como en el desarrollo de profármacos, aplicadas al campo de la senescencia celular. En el primer capítulo se introducen, a nivel general, los diferentes conceptos relacionados con el reconocimiento molecular y la detección de biomarcadores. Seguidamente se introducen conceptos básicos de senescencia celular y envejecimiento, así como el papel que juegan las células senescentes a nivel fisiológico, en enfermedades asociadas al envejecimiento celular y en el cáncer. Por último, se aborda también la creciente necesidad de desarrollar nuevas herramientas de diagnóstico y terapias centradas en la detección y tratamiento de dichas enfermedades. En el segundo capítulo se exponen los objetivos generales de la presente tesis doctoral, así como los objetivos concretos que son abordados en los diferentes capítulos experimentales. En el tercer capítulo se describe una nueva sonda molecular para la detección de senescencia in vivo. En concreto, se describe el diseño de una sonda de dos fotones basada en un fluoróforo de naftalimida conectada, mediante un éster metílico de L-histidina, con una galactosa acetilada que esta unida a uno de los átomos de nitrógeno aromático de la L-histidina a través de un enlace N-glicosídico hidrolizable (AHGa). La sonda inicial presenta una baja emsión pero en células senescentes se transforma en un fluoróforo con alto rendimiento cuantico que permite visualizar estas células debido a la hidrólisis del enlace N-glicosidico por la enzima ¿-galactosidasa sobreexpresada en este tipo de células. La sonda es capaz de detectar in vitro células de melanoma humano SK-Mel-103 tratadas con quimioterapia inductora de senescencia frente a células control. Adicionalmente, se validó la detección in vivo de senescencia en ratones con xenoinjertos tumorales tratados con quimioterapia inductora de senescencia. Basándonos en los resultados obtenidos en el capítulo tres, en el capítulo cuatro se describe un sistema similar cuyas características confieren al fluoróforo una mayor longitud de onda de excitación (HeckGal). La nueva sonda es capaz de detectar células senescentes debido a la sobreexpresión de la enzima ¿-galactosidasa asociada a senescencia en gran variedad de líneas celulares con diversos métodos de inducción de senescencia. Esta sonda se validó también in vivo en un modelo ortotópico de cáncer de mama de ratón tratado con quimioterapia inductora de senescencia, y en un modelo de ratón de fibrosis renal. Seguidamente, el capítulo cinco, se centra en un nuevo concepto de sondas moleculares no invasivas, que proporcionan una señal fácilmente legible a través de simples medidas de fluorescencia de la orina. Esta idea se llevó a cabo mediante la funcionalización de una sonda con grupos que le confieren características diuréticas (rápida eliminación renal) y el concepto se aplicó al diseño de una sonda para a la detección en orina de la carga senescente en diversos modelos. La sonda (Cy7Gal) está basada en la cianina-7 que es hidrolizada por la enzima ¿-galactosidasa en un fluoróforo altamente emisivo Cy7. Cy7Gal y Cy7 contienen restos de ácido sulfónico que aumentan su solubilidad en agua y desencadenan su rápida excreción por el sistema urinario. El grado de senescencia se cuantificó por medición directa de fluorescencia en orina en tres modelos de senescencia en ratones: (i) un modelo ortotópico de cáncer de mama en ratones BALB/cByJ con senescencia inducida por quimioterapia; (ii) ratones BALB / cByJ envejecidos de forma natural; y (iii) ratones con senescencia acelerada (SAMP8, del inglés Senescence Accelerated Mouse-Prone 8). Además, las imágenes IVIS ex vivo permite / [CA] La tesi doctoral titulada "Noves sondes, portadors i profàrmacs dirigits a cèl·lules senescents in vivo" se centra en el desenvolupament i avaluació de sondes basades en fluoròfors funcionalitzats o en nanopartícules, així com en el desenvolupament de profàrmacs, aplicades a el camp de la senescència cel·lular. Primer s'introdueixen els diferents conceptes relacionats amb el sensing i amb la sescencia cel·lular, abordant la necessitat de noves eines diagnòstiques i terapèutiques d'aquestes malalties. En els capítols 3:04 es descriuen una nova sonda molecular dos fotons per a la detecció de senescència in vivo en ratolins amb xenoempelts tumorals tractats amb quimioteràpia inductora de senescència, una altra sonda per a la detecció in vivo en un model ortotòpic de càncer de mama de ratolí tractat amb quimioteràpia inductora de senescència, i en un model de ratolí de fibrosi renal. El capítol 5, se centra en un nou concepte de sondes moleculars no invasives, que proporcionen un senyal fàcilment llegible a través de simples mesures de fluorescència de l'orina. Finalment, pel que fa a detecció de senescència cel·lular es refereix, es presenta en el capítol sis la detecció in vivo de senescència cel·lular mitjançant l'alliberament controlat de Nile Blue en nanopartícules de sílice mesoporoses funcionalitzades amb un galactohexasacárido. Un cop assolits els objectius planificats pel que fa a la diagnosi, en el capítol set es va abordar la millora d'un dels senolíticos més potents i àmpliament conegut en el camp de la senescència cel·lular que existeix a dia d'avui al mercat: el Navitoclax. / [EN] The doctoral thesis entitled "New probes, carriers and pro-drugs directed to senescent cells in vivo" focuses on the development and evaluation of probes based on functionalized fluorophores or nanoparticles, as well as on the development of prodrugs, applied to the field of cellular senescence. First, the different concepts related to sensing and cell sescence are introduced, addressing the need for new diagnostic and therapeutic tools for these diseases. Chapters three and four describe a new two-photon molecular probe for in vivo senescence detection in mice with tumor xenografts treated with senescence-inducing chemotherapy, another probe for in vivo detection in an orthotopic mouse breast cancer model. treated with senescence-inducing chemotherapy, and in a mouse model of renal fibrosis. Chapter five focuses on a new concept of non-invasive molecular probes, which provide an easily readable signal through simple measurements of urine fluorescence. Finally, regarding the detection of cellular senescence, chapter six presents the in vivo detection of cellular senescence through the controlled release of Nile Blue in mesoporous silica nanoparticles functionalized with a galactohexasaccharide. Once the planned objectives with regard to diagnosis had been achieved, chapter seven addressed the improvement of one of the most powerful and widely known senolytics in the field of cellular senescence that exists on the market today: Navitoclax. / The authors thank the financial support from the Spanish Government (projects MAT2015-64139-C4-1-R and AGL2015-70235-C2-2-R) and the Generalitat Valenciana (project PROMETEOII/2014/047). R.M laboratory members thank the financial support from the Spanish Government (project RTI2018-100910-B-C41) and the Generalitat Valenciana (project PROMETEO 2018/024). B.L-T. is grateful to the Spanish Ministry of Economy for their PhD grants (FPU15/02707). Work in the laboratory of MS was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), and the “laCaixa” Foundation. D.M.-E. laboratory is supported by the Cancer Research UK (CRUK) Cambridge Centre Early Detection Programme, by a CRUK Early Detection OHSU Project Award (C62187/A26989), by a Medical Research Council (MRC) New Investigators Research Grant (NIRG, MR/R000530/1). Work in the laboratory of I.F. is supported by grants from the Spanish Government (SAF2017-86690-R), Instituto de Salud Carlos III (CIBERNED and RETIC Tercel), and Generalitat Valenciana (Prometeo 2017/030). / Lozano Torres, B. (2021). Novel probes, carriers and prodrugs to target senescent cells in vivo [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172175

Doctor of Philosophy / Department of Physics / Itzhak Ben-Itzhak / Patrick Richard / This work focuses on the ultrafast dynamics of electrons and phonons in graphitic materials. In particular, we experimentally investigated the factors which influence the transport properties of graphite and carbon nanotubes. In the first part of this dissertation, we used Time-resolved Two Photon photoemission (TR-TPP) spectroscopy to probe the dynamics of optically excited charge carriers above the Fermi energy of double-wall carbon nanotubes (DWNTs). In the second part of this study, time-resolved anti-Stokes Raman (ASR) spectroscopy is applied to investigating in real time the phonon-phonon interactions, and addressing the way the temperature affects the dynamics of single-wall carbon nanotubes (SWNTs) and graphite. With respect to the first part, we aim to deeply understand the dynamics of the charge carriers and electron-phonon interactions, in order to achieve an as complete as possible knowledge of DWNTs. We measured the energy transfer rate from the electronic system to the lattice, and we observed a strong non-linear increase with the temperature of the electrons. In addition, we determined the electron-phonon coupling parameter, and the mean-free path of the electrons. The TR-TPP technique enables us to measure the above quantities without any electrical contacts, with the advantage of reducing the errors introduced by the metallic electrodes. The second investigation uses time-resolved ASR spectroscopy to probe in real time the G-mode non-equilibrium phonon dynamics and the energy relaxation paths towards the lattice by variation of the temperature in SWNTs and graphite. The lifetime range of the optically excited phonons obtained is 1.23 ps to 0.70 ps in the lowest (cryogenic temperatures) and highest temperature limits, respectively. We have also observed an increase in the energy of the G-mode optical phonons in graphite with the transient temperature. The findings of this study are important since the non-equilibrium phonon population has been invoked to explain the negative differential conductance and current saturation in high biased transport phenomena.

Neste trabalho, são estudadas as alterações das propriedades ópticas lineares e não lineares de soluções de azocompostos devidas aos mecanismos de isomerização e às alterações das propriedades estruturais com a temperatura. A dependência da transição pipi* com a estrutura linear dos azocompostos é notada já nas medidas dos espectros de absorção em função da temperatura para o isômero trans. Através do conhecimento dos níveis de energia e dos tempos de relaxação via isomerização, foi possível obter a dinâmica entre os isômeros cis e trans. As medidas não lineares forneceram propriedades interessantes associadas aos estados de energia dos compostos. Por exemplo, através de experimentos de varredura-z e de excitação e prova, foi possível ver que os azocompostos apresentam uma alta transparência quando excitados, a qual desaparece com o término da isomerização. Medidas em função do comprimento de onda mostraram que a transparência observada está presente em toda a banda pipi* e não ocorre na banda npi* . Também foi observado que o tempo de isomerização muda de acordo com o comprimento de onda de excitação, o que pode estar relacionado com a superposição das duas bandas. Os resultados obtidos em femtossegundos foram essenciais para descrever o processo dinâmico de fotoisomerização, observado, por completo, através de medidas com varredura-Z em picossegundos e nanossegundos. Essas medidas forneceram os valores das seções de choque dos isômeros cis, que são difíceis de serem quantificadas devido ao curto tempo de vida desse isômero. Além dos resultados ressonantes, foram feitos experimentos de absorção de dois fótons em femtossegundos usando a técnica de varredura-Z. Esses estudos mostraram a dependência das seções de choque de absorção de dois fótons com características estruturais das moléculas, tais como comprimento de conjugação, grupos push-pull e planaridade. Os resultados ressonantes e não ressonantes obtidos em femtossegundos serviram de base para a calibração da técnica de varredura-Z com pulsos de luz branca. Essa técnica se mostrou adequada para a obtenção dos espectros das não linearidades ópticas ressonantes e não ressonantes em uma única medição (de 5 minutos), diminuindo assim flutuações do laser durante o experimento. / This work reports on the temperature dependence of linear and nonlinear properties of azocompounds solutions due to isomerization mechanisms. The dependence of pipi* transitions on the linear structure of azocompounds is already noticeble in the measurements of absorption spectra as function of the temperature for the trans isomer. Knowing the energy levels and the relaxation times through isomerization, it was possible to obtain the exchange dynamics between cis and trans conformations. Nonlinear measurements provided interesting properties associated with the energy levels of de compounds. For exemple, through Z-scan and pumpprobe experiments, it was possible to verify that azocompounds present a high transparency when excited and that this transparency disapears when the izomerization ends. The wavelength change has shown that the observed transparency is present along the complete pipi* band, but not in the npi* band. It was also observed that the isomerization time changes with the exciting wavelength, which may be related to the superposition of both bands. The results obained with femtoseconds pulses were essential to completily describe the photoisomerization process observed with Z-scan measurements using picoseconds and nanoseconds pulses. These measurements provided values of the crosssection of the cis conformation, which are difficult to be quantified due to the short lifetime of this isomer. Besides ressonant results, experiments of two-photon absorption in the femtosecond regime using the Z-scan technique were made. These studies shown the dependence of the two photons absorption cross-section on structural features of molecules such as conjugation length, push-pull groups and planarity. The ressonant and nonressonant results obteined with femtoseconds have provide the calibration of the Z-scan technique with white light pulses. This technique has been found able to obtain the spectra of ressonat and nonressonant nonlinearities in a single measurement (about 5 minutes), diminishing laser fluctuation during the experiment.

Dendritic spines are fundamental units of information processing within the nervous system, responsible for independent modulation of synaptic input to neurons. Filopodia, often morphologically indistinguishable from spines, are involved in formation of synapses during neuronal development. Despite the importance of these structures for neuronal function, no detailed study of their presence on motoneurons has yet been made. Here, the presence of spines on hypoglossal motoneurons (HMs) is described at three developmental stages: at P0–2 and P9–11, spines are present at an average density of ~0.1 spines/micron, but at P19 spine density becomes negligible. In P0–2 and P9–11, spines are nonuniformly distributed, occuring in clusters, and at lower density in the most proximal and distal regions to the soma than at intermediate regions. HM spines coincide with a decrease in cell input resistance, which reduces excitability during development. Thus one may speculate that these spines are involved in the formation of new synapses required to maintain adequate excitatory drive. A major difficulty for the study of spines is their small size, which complicates measurement using optical methods. Here, I present a novel method for reconstructing spine morphology using geometric models based on a priori knowledge of spine structure. Tests of the technique using simulated data indicate that it has a resolving capability of up to 40 nm (limited by noise). The technique has been used to measure dendritic spines on HMs, showing that these structures have necks as small as 0.22 micron. For purely passive modulation of synaptic strength, spine necks need to be <~ 0.15 micron. These data suggest that if modulation of synaptic input occurs, biochemical and/or active electrical processes are needed. The methods developed in this Thesis, which have here been applied to HMs, are generally applicable to the study of spine morphology, and its effect on synaptic processing, in all classes of neurons.

S'ha estudiat la cinètica de fotosensibilització de l'oxigen singlet (1O2) en cèl·lules eucariotes en suspensió mitjançant un espectròmetre d'última generació amb resolució temporal per sota del microsegon. Els estudis revelen que la cinètica del 1O2 depèn del seu lloc de formació. Per una banda, la producció del 1O2 es més lenta en els lisosomes que en el nucli. Per altra banda, el 1O2 es capaç d'escapar de les cèl·lules quan es fotosensibilitza en el nucli, però es queda confinat al interior si es fotosensibilitza en els lisosomes. Malgrat que el temps de vida del 1O2 es troba en els microsegons, la desactivació principal ve donada per interaccions amb les biomolècules característiques de cadascú dels orgànuls. La incertesa respecte a la producció de 1O2 en un orgànul determinat es pot eliminar mitjançant l'ús de fotosensibilitzadors modificats genèticament, ja que aquets poden ésser expressats selectivament. Amb aquesta finalitat, s'avaluen les propietats fotosensibilitzants de mutants de proteïna fluorescent verd (GFP). Algunes de les GFPs estudiades sensibilitzen la formació del 1O2 malgrat amb baixa eficiència. Els resultats obtinguts es comparen amb els del cromòfor de la GFP i mostren que l'estructura proteínica, a sobre de modular les propietats fotofísiques del cromòfor, també el protegeix de la desactivació col·lisional. Finalment, s'estudien les propietats d'absorció bifotónica del 2,7,12,17-tetrafenilporficé i del seu complex de pal·ladi (II). L'eficiència de formació del 1O2 per part dels dos compostos, desprès de l'absorció simultània de dos fotons, es aproximadament 100 vegades superior a la dels seus anàlegs porfirínics, amb seccions d'absorció bifotòniques &#948; ~ 25 GM. Les excel·lents propietats d'aquestos compostos s'expliquen mitjançant arguments qualitatius i s'analitzen les seves perspectives de cara al seu ús en teràpia fotodinámica. / Se ha estudiado la cinética de fotosensibilización de 1O2 en células eucariotas en suspensión, usando un espectrómetro de última generación con resolución temporal por debajo del microsegundo. Los estudios revelan que la cinética del 1O2 depende de su lugar de formación. Por una parte, la producción de 1O2 es más lenta en los lisosomas que en el núcleo. Por otra parte, el 1O2 es capaz de escapar de las células cuando es fotosensibilizado en el núcleo, mientras que queda confinado en el interior si se fotosensibiliza en los lisosomas. A pesar de que el tiempo de vida del 1O2 se encuentra en los microsegundos, la desactivación principal viene dada por interacciones con las biomoléculas características de cada orgánulo. La incertidumbre respecto a la producción de 1O2 en un orgánulo determinado puede ser eliminada mediante el uso de fotosensibilizadores modificados genéticamente ya que pueden ser expresados selectivamente. Con este fin, se evalúan las propiedades fotosensibilizantes de mutantes de proteína fluorescente verde (GFP). Algunas de las GFPs estudiadas sensibilizan la formación de 1O2 aunque con baja eficiencia. Los resultados obtenidos se comparan con los del cromóforo de la GFP y muestran que la estructura proteínica, además de modular las propiedades fotofísicas del cromofóro, también lo protege de la desactivación colisional. Finalmente, se estudian las propiedades de absorción bifotónica del 2,7,12,17-tetrafenilporficeno y de su complejo de paladio (II). La eficacia de formación de 1O2 de ambos compuestos, tras la absorción simultánea de dos fotones, es aproximadamente 100 veces superior a la de sus análogos porfirínicos, con secciones de absorción bifotónica &#948; ~ 25 GM. Las excelentes propiedades de estos compuestos se explican mediante argumentos cualitativos y se analizan sus perspectivas de cara a su uso en terapia fotodinámica. / The kinetics of singlet oxygen (1O2) photosensitisation in human skin fibroblasts have been investigated by means of an ultrasensitive near-infrared spectrometer with submicrosecond time resolution. The results indicate that the 1O2 kinetics are site-dependent. On one hand, the production of 1O2 is slower in the lysosomes than in the nucleus. On the other hand, 1O2 is able to escape out of the cells when photosensitised in the nucleus, while 1O2 photosensitized in the lysosomes is confined. Despite showing a lifetime in the microsecond time domain, the decay of 1O2 is governed by interactions with the biomolecules within the organelle there it is produced. The uncertainty as to the intracellular site of 1O2 production may be removed by the use of genetically-encoded photosensitisers, which can be expressed in any desired organelle. Towards this end, the ability of some fluorescent proteins (GFPs) to photosensitise 1O2 has been studied. Some of the studied proteins are able to produce 1O2 albeit with a very low quantum yield. The results obtained are compared to those of the synthetic GFP chromophore and indicates that the protein scaffold not only plays a role in modulating the photophysical properties of the chromophore but also has a protective function from collisional quenching. Finally, the two-photon absorption properties of tetraphenylporphycene and its palladium (II) complex have been determined. These compounds are ca. 100-fold more efficient two-photon 1O2 photosensitisers than their isomeric porphyrin counterparts, with two-photon absorption cross sections &#948; ~ 25 GM. Qualitative symmetry-based arguments are provided to explain the excellent two-photon properties and the prospects for photodynamic therapy are discussed.

Cancer is a leading cause of death that affects millions of people across the globe each year. Photodynamic therapy (PDT) is a relatively new treatment approach for cancer in which anticancer drugs are activated by light at an appropriate wavelength to generate highly cytotoxic reactive oxygen species (ROS) and achieve tumor destruction. Compared with conventional chemo- and radiotherapy, PDT can be performed with minimal invasiveness, local targeting and reduced side effects. However, most of the currently available PDT drugs mainly absorb in the visible part of the spectrum, where light penetration depth into human tissues is very limited. Therefore, increasing the treatment depth of PDT has been considered to be an important approach to improve the effectiveness of PDT for treating larger and thicker tumor masses. In this thesis, we present our investigation into the potential of two-photon activated PDT (2-γ PDT), combination therapy of PDT and chemotherapy, and bioluminescence-activated PDT as a means to increase the treatment depth of this modality. In 2-γ PDT, the photosensitizing agents are activated through simultaneous absorption of two photons. This approach allows the use of near-infrared (NIR) light that can penetrate deeper into tissues and thus, has the potential of treating deep-seated tumors and reducing side effects, while the non-linear nature of two-photon excitation (TPE) may improve tumor targeting. We have evaluated the PDT efficacy of a second-generation photosensitizer derived from chlorophyll a, pyropheophorbide a methyl ester (MPPa), through both one- and two-photon activation. We observed that MPPa had high one-photon (1-γ PDT efficacy against both cisplatin-sensitive human cervical (HeLa) and cisplatin-resistant human lung (A549) and ovarian (NIH:OVCAR-3) cancer cells when activated by femtosecond (fs) laser pulses at 674 nm. At a low light dose of 0.06 J cm-2, the MPPa concentration required to produce a 50% cell killing effect (IC50) was determined to be 5.3 ± 0.3, 3.4 ± 0.3 and 3.6 ± 0.4 μM in HeLa, A549 and NIH:OVCAR-3 cells, respectively. More significantly, we also found that MPPa could be effectively activated at the optimal tissue-penetrating wavelength of 800 nm through TPE. At a light dose of 886 J cm-2, where no measurable photodamage was observed in the absence of MPPa, the IC50 values were measured to be 4.1 ± 0.3, 9.6 ± 1.0 and 1.6 ± 0.3 μM in HeLa, A549 and NIH:OVCAR-3 cells, respectively. We obtained corresponding LD50 (the light dose required to produce a 50% killing effect) values of 576 ± 13, 478 ± 18 and 360 ± 16 J cm-2 for 10 μM MPPa, which were approximately 3-5 times lower than the published 2-γ LD50 of Visudyne® and 20-30 times lower than that of Photofrin®. These results indicate that MPPa may serve as a photosensitizer for both 1- and 2-γ activated PDT treatment of difficult-to-treat tumors by conventional therapies. Indocyanine green (ICG), a dye having an absorption maximum near 800 nm, has been considered to be a potential NIR PDT agent. However, the PDT efficacy of ICG has been found to be very limited probably due to the low yield of cytotoxic ROS. In the present work, we have evaluated the combination effects of ICG-mediated PDT with conventional chemotherapy mediated by two types of chemotherapeutic drugs, namely the type II topoisomerase (TOPII) poisons etoposide (VP-16)/teniposide (VM-26) and the platinum-based drugs cisplatin (CDDP)/oxaliplatin (OXP). Synergistic enhancement of cytotoxicity and increased yields of DNA double strand breaks (DSBs) were observed in HeLa, A549 and NIH:OVCAR-3 cancer cells treated with the combination of ICG-PDT and VP-16. The presence of VP-16 during the laser irradiation process was found to be critical for producing a synergistic effect. An electron-transfer-based mechanism, in which ICG could increase the yield of highly cytotoxic VP-16 metabolites, was proposed for the observed synergistic effects, although direct spectroscopic detection of the reaction products was found to be very challenging. Moreover, we observed a much lower degree of synergy in the human normal fibroblast GM05757 cells than that in the three cancer cell lines investigated. Synergistic effects were also observed in A549 cells treated with the combination of ICG-PDT and VM-26 (i.e. an analog of VP-16). Furthermore, the combination of low-dose CDDP/OXP and ICG-PDT was demonstrated to produce an additive or synergistic effect in selected cancer cell lines. These preliminary results suggest that the combination of ICG-PDT with VP-16/VM-26 or CDDP/OXP chemotherapy may offer the advantages of enhancing the therapeutic effectiveness of ICG-PDT and lowering the side effects associated with the chemotherapeutic drugs. Bioluminescence, the generation of light in living organisms through chemical reactions, has been explored as an internal light source for PDT in recent years. This approach, in principle, does not suffer from the limited tissue penetration depth of light. In the present project, we have evaluated the effectiveness of luminol bioluminescence in activating the porphyrin photosensitizers meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TPPS4) and Fe(III) meso-tetra(4-sulfonatophenyl)porphine chloride (FeTPPS). The combination treatment induced significant killing of HeLa cells, while additive effects were observed in two normal human fibroblast cell lines (GM05757 and MRC-5). Our observations indicate that bioluminescence of luminol may generate sufficient light for intracellular activation of PDT sensitizers. Furthermore, the combination treatment may have intrinsic selectivity towards cancerous tissues. In summary, we have demonstrated effective killing of cancer cells by MPPa-mediated 1- and 2-γ PDT, combination of ICG-PDT and VP-16/VM-26 or CDDP/OXP chemotherapy, and bioluminescence of luminol activated PDT mediated by TPPS4/FeTPPS. These positive preliminary results indicate that all these three approaches have the potential of increasing the treatment depth of PDT and facilitating the development of more effective PDT treatment strategies.