The two main themes in this thesis are the implementation of interference methods with optically trapped particles for measurements of position and optical phase (optical interferometric metrology) and the optical manipulation of nanoparticles for studies in the assembly of nanostructures, nanoscale heating and nonlinear optics (nanophotonics). The first part of the thesis (chapter 1, 2) provides an introductory overview to optical trapping and describes the basic experimental instrument used in the thesis respectively. The second part of the thesis (chapters 3 to 5) investigates the use of optical interferometric patterns of the diffracting light fields from optically trapped microparticles for three types of measurements: calibrating particle positions in an optical trap, determining the stiffness of an optical trap and measuring the change in phase or coherence of a given light field. The third part of the thesis (chapters 6 to 8) studies the interactions between optical traps and nanoparticles in three separate experiments: the optical manipulation of dielectric enhanced semiconductor nanoparticles, heating of optically trapped gold nanoparticles and collective optical response from an ensemble of optically trapped dielectric nanoparticles.

The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW₃ proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.

De nouveaux nano-objets anisotropes fluorescents sont obtenus par l’assemblage d’acènes spécifiquement conçus. Dans des cristaux, nano-rubans et nanoparticules anisotropes de 2,3-dialkyldiphenylanthracènes, les efficacités et la polarisation de l’émission bleue sont remarquables. La couleur de l’émission est accordée par le dopage avec des émetteurs verts et oranges (di- et tétra-phényltétracènes). La microscopie confocale de fluorescence permet d’étudier les cinétiques des états excités et des transferts d’énergie photo-induits, ainsi que la dispersion et les orientations des émetteurs. Pour la première fois, l’influence de la largeur de nano-rubans sur la cinétique d’annihilations triplet-triplet de tétracènes est mise en évidence. La microscopie révèle également le polymorphisme inhabituel d’un dérivé diéthynylphényl-anthracène. Ce travail ouvre des perspectives pour le développement et l’étude de processus fondamentaux de nano-matériaux luminescents. / New fluorescent anisotropic nano-objects are obtained by the assembly of specifically designed acenes. In crystals, nano-ribbons and anisotropic nanoparticles of 2,3-dialkyldiphenylanthracenes, the efficiencies and the polarization of the blue emission is remarkable. The color of the emission is tuned by doping with green and orange emitters (di-and tetra-phenyltetracenes). Confocal fluorescence microscopy is used to study the kinetics of excited states and photo-induced energy transfers, as well as the dispersion and orientation of the emitters. For the first time, the influence of the width of the nano-ribbons on the kinetics of tetracene triplet-triplet annihilations is highlighted. Microscopy also reveals the unusual polymorphism of a diethynylphenyl anthracene derivative. This work opens perspectives for the development and study of fundamental processes of luminescent nano-materials.

Gene regulation by transcription factors are one of the key processes that are important to sustain all kinds of life. In the prokaryote Escherichia coli this has shown to especially crucial. The operator sequence to which these transcription factors bind to are very small in comparison to the whole genome of E. coli, thus the question becomes how these proteins can find these sequences quickly. One particularly well-studied transcription factor in this regard is the lac repressor. It has been shown that this transcription factors finds its operators faster than the limit of three dimensional diffusion. The leading model for how the repressor does that is facilitated diffusion and this model has gained more experimental evidence, particularly using single-molecule fluorescence microscopy. This study aimed at measuring the unspecific binding time between the lac repressor and DNA in vivo, but in the end the project evolved to trying to establish a single-molecule tracking assay of the repressor in vivo. In this study a mutant of the repressor was expressed and purified, labelled with a synthetic fluorophore, electroporated into E. coli and tracking was performed under a microscope. One of the three types of experiments were partially analysed with an image analysis software. Unfortunately, analysis was not completed for all experiments which made it difficult to compare the results. In the end the data was compared by eye while also using the results from image analysis. With slight optimism it can be concluded that the assay worked, but it needs more development.

Introdução. O transplante de células é possibilidade terapêutica promissora para muitas doenças neurológicas. Nos últimos anos, a possibilidade do isolamento de células-tronco dos tecidos adultos, por exemplo da medula-óssea, atrai a atenção da comunidade científica, estratégia que minimiza os problemas éticos relativos ao uso de tecido fetal para implantes visando ao tratamento de doenças neurológicas. Entretanto, a eficiência da transdiferenciação de células-tronco mesenquimais em neurônios, bem como os mecanismos envolvidos nesse processo, permanecem desconhecidos. A obtenção de neurônios maduros ocorreu somente em sistemas de co-cultura, o que induz a questão se a diferenciação representa um potencial das células per si, ou se é possível somente devido à fusão com neurônios maduros. Objetivos. No presente trabalho, pretendeu-se verificar o potencial de as células-tronco mesenquimais tornarem-se neurônios e esclarecer os possíveis mecanismos envolvidos nesse processo. Material e métodos. Células-tronco mesenquimais foram isoladas de 20 doadores voluntários normais e caracterizadas por análise de separação celular ativada por fluorescência. A multipotencialidade foi investigada ao se diferenciar as células em condrócitos e osteócitos. A capacidade de auto-renovação foi confirmada pelo ensaio de incorporação de BrdU. Ulteriormente, as células foram diferenciadas por uma semana em meio contendo AMPc, IBMX, ou combinação de ambos, e os resultados foram comparados com o cultivo em meio básico. Diferentes bloqueadores de Ca2+ ou inibidores de PKA foram usados como tentativa de se impedir a diferenciação, ocorrência que foi mensurada com imunocitoquímica para NF-200 (marcador de neurônios maduros). O registro eletrofisiológico por meio de patch clamp foi usado para se confirmar o fenótipo neuronal. As figuras foram configuradas em microscopia confocal. Para análise estatística foi utilizada ANOVA com teste post-hoc. Resultados. As células isoladas expressaram CD90, 105, 44 e 13 mas foram negativas para CD34 e 45. Isto significa que não são de origem hematopoiética; 98,74 ± 0,43% das células incorporaram BrdU em 24 horas. Após o isolamento, foi possível diferenciá-las em condrócitos ou osteócitos. Em situação controle, não foram evidenciadas células positivas para NF200. Por outro lado, ocorreu positividade em 10,75% ± 1,35 (p<0,0001) das células sob IBMX e, em 15,18% ± 1,12, sob a combinação cAMP e IBMX (p<0,0001). Foram registradas correntes de Na+ e K+ dependentes de voltagem, mas não potenciais de ação. A diferenciação foi inibida com PKAi (5,73% ± 0,42, p<0,0001), nifedipina (5,79% ± 0,98, p<0,0001), Ni2+ (7,06% ± 1,68, p<0,0001) e Cd2+ (0 ± 0, p<0,0001). Discussão. Isolou-se uma população de células-tronco estromais da medula-óssea de seres humanos que se mostrou multipotencial e auto-renovável. O aumento da concentração de AMPc no meio elevou a concentração de neurônios para 15%. A diferenciação parece depender da via PKA mas também envolve a concentração intracelular de Ca2+. Conclusão. O correto entendimento de como as células-tronco mesenquimais diferenciam-se pode contribuir para aumentar a eficácia do método e, talvez um dia, tornar possível o uso dessa ferramenta no campo clínico. / Introduction. Cell transplantation has been considered a promising therapeutic approach for many neurological diseases. The possibility of isolation of stem cells from adult tissues, i.e. bone marrow, has attracted the attention of the scientific community in the recent years. This strategy is interesting on avoiding the ethical issues regarding the use of fetal tissue for neural implants. Moreover, the efficiency of the transdifferentiation of mesenchymal stem cells (MSCs) into neurons, and the mechanisms involved in this process remain largely unknown. The obtention of mature neurons was described only in coculture systems, what raised the question if the differentiation is a potential of the cells itself, or if it is possible only due to fusion with mature neurons. Objectives. In the present investigation, we aimed to verify the potential of MSCs to differentiate into neurons, and also to clarify the possible mechanisms involved on it. Material and methods. MSCs were isolated from 20 healthy human subjects and characterized by FACS-analysis. Multipotentiality was addressed by differentiating them into chondrocytes and osteocytes. The self-renewal capacity was confirmed with BrdU-incorporation assay. Afterwards, cells were differentiated for 1 week in a medium containing cAMP, IBMX, or a combination of both, and the results were compared with cells treated in basal-medium condition. Different Ca2+-blockers and PKA-inhibitor peptide were used on an attempt to impair differentiation, which was quantified with NF-200 immunostaining (a marker of mature neurons). Patch-clamp recording was used to confirm neuronal phenotype. Pictures were taken in confocal microscope. For statistical analysis ANOVA with a post-hoc test was used. Results. The isolated cells expressed CD90, 105, 44, and 13, but were negative for CD34 and 45, meaning that they were non-hematopoiethic; 98.74 ± 0.43 % of them incorporated BrdU in 6hs. After isolation, they differentiated into chondrocytes and osteocytes. In a control situation, no NF200 positive cell was seen. On the other hand, 10.75% ± 1.35 (p<.0001) of positivity was seen under IBMX and 15.18% ± 1.12 in the combination of cAMP with IBMX (p<.0001). Na+ and K+-voltage gated currents were recorded. Differentiation was impaired with PKAi (5.73% ± 0.42, p<.0001), nifedipin (5.79% ± 0.98, p<.0001), Ni2+ (7.06% ± 1.68, p<.0001), and Cd2+ (0 ± 0, p<.0001). Discussion. We were able to isolate a population of stromal stem cells from the bone marrow of human subjects, since they were multipotential and self-renewable. Increasing the concentration of cAMP raised the percentage of neurons up to 15%. The differentiation seems to be dependent on the PKA pathway, but also involved the intracellular concentration of Ca2+. Conclusions. The complete understanding of how MSC differentiate can contribute to increase the efficiency of the method and thus make possible to use this powerful tool in the clinical practice.

O objetivo do trabalho foi determinar o pHi, a capacidade tamponante do citosol na ausência de CO2/HCO3- (&#946;i) de neurônios dos gânglios das raízes dorsais e investigar a expressão de trocadores Na+/H+ e sua função na regulação do pHi. O pHi foi estimado com o indicador fluorescente BCECF. A operação do trocador era quantificada na recuperação da acidose intracelular induzida experimentalmente. Na ausência do tampão CO2/HCO3- a taxa de alcalinização (k) deve-se, por hipótese, ao transporte de H+ pelo trocador. A hipótese foi confirmada pela ação de agentes farmacológicos, e.g., amiloride. Em soluções tamponadas por CO2/HCO3- as células tem pHi de 7,24 e, em soluções tamponadas com HEPES, 7,04. A &#946;i foi de 8,17 mM/pH. As células se recuperam da acidose com k médio de 0,0138 s-1. O efeito inibitório do amiloride em concentração de 1 mM deve-se ao fato dos fenótipos celulares expressarem diferentes isoformas do trocador. Segundo RT-PCR, todas as 5 isoformas do trocador são expressas e a quantidade de RNAm, avaliada por qPCR, é maior para a NHE1, seguida de NHE5. / The objective here was to determine intracellular pH (pHi), cytosolic buffering power in CO2/HCO3- free medium (&#946;i) of neurons from dorsal root ganglia and to investigate the functional expression of the Na+/H+ exchangers in the regulation of pHi. pHi was estimated with fluorescence indicator BCECF. Exchanger operation was quantified during recovery from intracellular acidification induced experimentally. In CO2/HCO3- free medium the alkalinization rate (k) is due, hypothetically, H+ extrusion by the exchanger. This assumption was confirmed by action of pharmacologic agents, e.g., amiloride. In medium buffered with CO2/HCO3- cells have pHi of 7.24 and, in medium buffered with HEPES, 7.04. &#946;i calculated was 8.17 mM/pH. Cells recovery from acidosis with mean k of 0.0138 s-1. Inhibitory effect of amiloride in 1 mM concentration is due to cellular phenotypes expressing different Na+/H+ exchanger isoforms. According to RT-PCR, all the five exchanger isoforms are expressed and mRNA quantity, evaluated by qPCR, is greater to NHE1, followed by NHE5.

A resposta do sistema nervoso para diversas lesões acarreta a ativação das células gliais entéricas. Este trabalho tem como objetivo analisar o efeito da isquemia e reperfusão intestinal (I/R-i) sobre as células gliais entéricas, neurônios e receptores P2X2 e P2X7. Foram analisados o íleo de ratos Controle, Sham e I/R-i com 0 hora, 24 horas e 14 dias de reperfusão. Foram realizadas dupla marcação dos receptores P2X2 e P2X7 com Hu e S100, densidade, área do perfil e marcação de proliferação celular. Os resultados mostraram dupla marcação de células gliais entéricas e neurônios com os receptores P2X2 e P2X7; a densidade apresentou um aumento de células gliais e diminuição de neurônios imunorreativos ao Hu. A área do perfil de células gliais entericas S100-IR apresentaram diminuição nos grupos I/R-i e foi detectada proliferação de células gliais entéricas nos grupos I/R-i 0 hora e 24 horas. Conclui-se que a isquemia levou a alterações diferenciadas nos receptores P2X2 e P2X7, células gliais entéricas e neurônios, que podem causar disfunções gastrointestinais. / The nervous system response to various injuries involves the activation of enteric glial cells. The aim of the work was to analyze the effect of ischemia and reperfusion (I/R-i) on enteric glial cells, neurons and receptors P2X2 and P2X7. We analyzed the ileum of Control, Sham and I/R-i with 0 hour, 24 hours and 14 days of reperfusion. Double staining were performed P2X2 and P2X7 receptors with Hu and S100, density, area profile and marking of cellular proliferation. The results show double staining of neurons and enteric glial cell with the P2X2 and P2X7; density increased by glial cells and decrease of neurons immunoreactive to Hu. The area profile of enteric glial cell S100-IR showed decreased in Groups I/R-I and enteric glial cell proliferation was observed in groups I/R-i 0 hours and 24 hours. It is concluded that ischemia has led to changes in differential P2X2 and P2X7 receptors, neurons and enteric glial cells, which can cause gastrointestinal dysfunction.

Low bioavailability of phosphate (P) due to low concentration and high immobility in soils is a key limiting factor in crop production. Application of excess amounts of P fertilizer is costly and by no means sustainable, as world-wide P resources are finite and running out. To facilitate the breeding of crops adapted to low-input soils, it is essential to understand the consequences of P deficiency. The second messenger calcium (Ca2+) is known to signal in plant development and stress perception, and most recently its direct role in signalling nutrient availability and deficiency has been partially elucidated. The use of Ca2+ as a signal has to be tightly controlled, as Ca2+ easily complexes with P groups and therefore is highly toxic to cellular P metabolism. It is unknown whether Ca2+ signals P availability or whether signalling is altered under P starvation conditions. The aim of this PhD project was to characterise the use of Ca2+ ions, particularly cytosolic free Ca2+ ([Ca2+]cyt), in stress signalling by P-starved roots of the model plant Arabidopsis thaliana. The hypothesis was that under P starvation and a resulting decreased cellular P pool, the use of [Ca2+]cyt may have to be restricted to avoid cytotoxic complexation of Ca2+ with limited P groups. Employing a range of genetically encoded Ca2+ reporters in Arabidopsis, P starvation but not nitrogen starvation was found to strongly dampen the root [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The strongly altered root [Ca2+]cyt response to extracellular nucleotides was shown to manifest itself during seedling development under chronic P deprivation, but could be reversed by P resupply. Fluorescent imaging elucidated that P-starved roots showed a normal [Ca2+]cyt response to extracellular nucleotides at the apex, but a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species (ROS) levels induced by P starvation. Excluding iron, as well as P, rescued the altered [Ca2+]cyt response, and restored ROS levels to those seen under nutrient-replete conditions. P availability was not signalled through [Ca2+]cyt. In another part of this PhD project, a library of 77 putative Ca2+ channel mutants was compiled and screened for aberrant root hair growth under P starvation conditions. No mutant line showed aberrant root hair growth. These results indicate that P starvation strongly affects stress-induced [Ca2+]cyt modulations. The data generated in this thesis further understanding of how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

En médecine, les nanotechnologies permettent le développement de nouvelles techniques de soin comme l’hyperthermie local ou la délivrance ciblée de médicaments. Ces applications impliquent de nouveaux défis scientifiques concernant la conception de nanosystems et les propriétés de leur environnement biologique. Dans cette thèse, nous avons analysé plusieurs aspects de la relaxation thermique de tels systèmes. Nous avons mise en œuvre la fois des simulations de Dynamique Moléculaire et des mesures expérimentales de microscopie d’imagerie en temps de vie de fluorescence. Nous présentons une étude numérique du transfert thermique depuis une nanoparticule en solution aqueuse et montrons qu’attacher un polymère à sa surface permet de réduire la résistance thermique entre la particule et son environnement. Nous avons modélisé des bicouches lipidiques pour calculer leurs propriétés diélectriques et leur viscosité a été étudiée par microscopie de fluorescence. Ces expériences sont réalisées sur des membranes suspendues et des vésicules unilamellaires géantes et démontrent que la viscosité des bicouches lipidiques diminue avec la température et l’application d’une tension transmembranaire induisant un changement de structure. / In medicine, nanotechnologies give the opportunity to create new care practices such as local hyperthermia and targeted drug delivery. These applications imply new scientific challenges concerning the design of nanodevices and the properties of their biological environment. In this thesis, we have analysed several aspects of heat relaxation of such systems. We have used both Molecular Dynamics numerical simulations and Fluorescence-lifetime imaging microscopy experiments. We present a study of heat transfer from a solvated nanoparticle and show that attaching a polymer on its surface reduces the thermal resistance between the particle and its aqueous environment. We have modelled lipid bilayers to compute their dielectric properties and their viscosity have been investigated by fluorescence imaging. The experiments conducted on both suspended lipid membrane and giant unilamellar vesicles show that the viscosity decreases when the temperature increases and when a transmembrane voltage is applied to inducing a structural change.

A terapia fotodinâmica (TFD) utilizando ácido 5-aminolevulinico (ALA) e derivados em aplicação tópica e, como precursor da protoporfirina IX (PPIX) apresenta alguns limitantes relativos a baixa permeação das substâncias na pele. Comportamento este que afeta a produção e homogeneidade da distribuição da PPIX na superfície e camadas mais profundas da pele. Para resolver essa limitação muitos autores propõem alternativas modificando a molécula do ALA e derivados, bem como modificando as propriedades químicas da fase externa da emulsão (mais hidrofílica ou hidrofóbica) ou então o sistema de entrega para a emulsão. O objetivo desse estudo é avaliar qual a proporção de ALA e metil-5-aminolevulinato (MAL) que quando misturados levam ao aumento da quantidade e uniformidade da formação da PPIX na superfície e em profundidade na pele. Para esse estudo foi realizada análises de fluorescência e histologia. O estudo foi conduzido in vivo e ex vivo usando biópsias de pele de porco cultivadas in vitro. A produção de PPIX foi monitorada utilizando espectroscopia de fluorescência, imagem de fluorescência de campo amplo e microscopia confocal de fluorescência. E para a aplicação da TFD os parâmetros usados foram de 125 mW/cm2 de intensidade e 150 J/cm2 de dose. A análise do dano causado pela irradiação foi realizada por meio de histologia da pele após 24 e 48 horas da aplicação da TFD. O ALA e MAL na concentração de 20% foram misturados nas seguintes proporções: ALA ou M, M2 (80% ALA - 20% MAL), M3 (60% ALA 40% MAL), M4 (50% ALA MAL), M5 (40% ALA 60% MAL), M6 (20% ALA 80% MAL) e MAL como M7. As diferentes proporções foram incorporadas em emulsões óleo em água (O/A) e água em óleo (A/O). De acordo com os resultados, as misturas M3, M4 e M5 mostraram maior produção de PPIX na superfície da pele segundo as medidas de fluorescência em 3h de incubação e, no estudo da cinética mostraram produzir PPIX em menor tempo. No estudo de permeação do creme in vitro em pele ex vivo, por microscopia confocal de fluorescência, observou-se que as misturas M3, M4 e M5 produziram mais PPIX nas camadas da pele do que ALA e MAL. As análises histológicas das misturas apresentaram maior dano fotodinâmico na superfície e profundidade das camadas da pele após a TFD, independente da emulsão. A análise em até 48h observou-se predominantemente a fase do processo de reparo referente à fase inflamatória, mas existem indícios ao longo das análises tanto macroscópicas e histológicas que o processo de reparo referente as fases subsequentes de proliferação e remodelamento estão iniciando-se em paralelo. A mistura M4 em ambas as emulsões apresentou elevada quantidade de formação de PPIX em menor tempo de incubação. M4 em emulsão O/A apresentou menor dano fotodinâmico já que a evolução do processo reparo foi mais rápida sugerindo-se potencial de aplicação em TFD voltado para área cosmética-estética. Já M4 em emulsão A/O levou a um maior dano fotodinâmico já que a evolução do processo de reparo foi mais lenta sugerindo-se potencial de aplicação em TFD voltado para área oncológica e de doenças de pele. De modo geral o estudo proposto apresentou impacto positivo para a otimização da terapia fotodinâmica em aplicação tópica. / Photodynamic therapy (PDT) using 5-aminolevulinic acid and derivatives on topical application and as a precursor of protoporphyrin (PPIX) has some limitations for low permeation of substances into the skin. This behavior affects PPIX production and homogeneous distribution on the surface and deeper layers of the skin. To resolve this limitation, many authors propose alternatives such as modifying the molecule of ALA and its derivatives, as well as changing the chemical properties of the external phase of the emulsion (more hydrophilic or hydrophobic) or the delivery system to the emulsion. The aim of this study is to assess the proportion of ALA and methyl-5-aminolevulinate (MAL) that when mixed leads to an increase in the amount and uniformity of the PPIX formation on surface and deep skin. For this study we performed fluorescence analysis and histology. The studies were conducted in vivo and also using pig skin biopsies (ex vivo) cultured in vitro. The PPIX production was monitored using fluorescence spectroscopy, widefield fluorescence imaging, and fluorescence confocal microscopy. For the application of PDT an intensity of 125 mW/cm2 and a dose 150 J/cm2 were used. Analysis of the damage caused by irradiation was performed through skin histology after 24 and 48 hours after PDT application. ALA and MAL in concentration of 20% were mixed in the following proportions: ALA or M, M2 (80% ALA - 20% MAL), M3 (60% ALA - 40% MAL), M4 (50% ALA - MAL) M5 (40% ALA - 60% MAL), M6 (20% ALA - 80% MAL) MAL and as M7. Different proportions were incorporated in oil-in-water emulsions (O/W) and water-in-oil (W/O). The fluorescence measurements for 3h of incubation showed better PPIX production in the skin surface for mixtures M3, M4 and M5. Moreover, the kinetics study showed PPIX production in less time for these mixtures. In the study of cream permeation of ex vivo skin in vitro by confocal fluorescence microscopy, we observed that the mixtures M3, M4 and M5 produced more PPIX in the skin layers than ALA and MAL. The histological analyses of the mixtures showed higher photodynamic damage on the surface and deeper layers of the skin after PDT, independent of the emulsion. The analysis in 48 hours predominantly observed the phase of the healing process regarding the inflammatory phase but there are signs along both macroscopic and histological analysis that the healing process concerning the subsequent stages of proliferation and remodeling are initiating in parallel. The mixture M4 in both emulsions had high amounts of PPIX formation in shorter incubation time. M4 emulsion O/A showed a lower photodynamic damage since the evolution of the healing process was faster suggesting to potential application in PDT facing cosmetic-aesthetic area. M4 already in W/O emulsion led to a greater photodynamic damage since the evolution of the healing process was slower suggesting to potential application in PDT facing oncology and skin diseases. Overall the proposed study had a positive impact on the optimization of photodynamic therapy for topical application.