Fluorescence based microscopy techniques have been extensively used in biological sciences. The most common approach is the steady-state fluorescence microscopy. Although the said approach is powerful, it often lacks sensitivity to detect several biochemical processes that may indicate relevant conditions of biological tissues. The fluorescence dynamics analysis not only brings intrinsic information about the tissue, but is also less sensitive to the medium scattering and absorption, and sometimes capable of distinguishing between fluorescent structures with indistinguishable spectra. The intrinsic fluorescence lifetime of biological tissues is usually affected by some clinical conditions, especially when those conditions cause or are correlated with metabolic modifications. Time-resolved spectroscopy techniques can be used to detect those modifications and may be used as a tool to improve the detection and diagnosis rate of such conditions. Fluorescence Lifetime Imaging Microscopy (FLIM) combines the temporal resolution and the microscopy concept, so fluorescence lifetime images can be generated. This technique has a great potential for clinical applications since it may be able to detected lesions and delineate its borders. However, FLIM usually demands a more sophisticated instrumentation than most techniques based on the steady-state approach, what creates a difficulty for moving such a system to a clinical setting. We report the assembly, characterization, validation, and clinical application of a multispectral FLIM system featuring a handheld probe composed of a laser scanning rigid endoscope. The assembled system uses a 355 nm short pulsed laser as excitation and has three spectral channels, targeting the emission of collagen, NADH, and FAD, which are important endogenous fluorophores. The system acquires images of 8.65 x 8.65 mm2 areas in ~ 2.4 s. MATLAB codes were written to process the images using a biexponential model and a modified phasor approach. In vivo validation measurements of tumors induced in mice were performed. The system was also validated with in vivo imaging of skin of healthy volunteers. The assembled FLIM system was moved to Hospital Amaral Carvalho, where we performed a pilot clinical study, in which different types of skin lesions were imaged in vivo in a clinical setting. A significant contrast was achieved on seborrheic keratosis, Bowen´s disease, and sclerodermiform basal cell carcinoma tumors. These results indicate the potential of this technique for clinical imaging of skin lesions. / Técnicas de microscopia baseadas em fluorescência têm sido extensamente utilizadas em ciências biológicas. A abordagem mais comum se baseia na microscopia de fluorescência de estado estacionário. Apesar de poderosa, essa abordagem frequentemente não apresenta sensibilidade suficiente para detectar diversos processos bioquímicos que podem ser indicadores de relevantes problemas em tecidos biológicos. A análise da dinâmica da fluorescência não apenas trás informações intrínsecas sobre o tecido, mas também é menos sensível a espalhamento e absorção pelo meio, além de ser capaz de distinguir entre estruturas fluorescentes com espectros indistinguíveis em alguns casos. O tempo de vida intrínseco de tecidos biológicos é normalmente afetado por condições clínicas, especialmente quando estas condições causam ou são relacionadas a modificações metabólicas. As técnicas de espectroscopia resolvidas no tempo podem detectar essas modificações e podem ser utilizadas como uma ferramenta para melhorar a detecção e o diagnóstico dessas condições. A Microscopia de Tempo de Vida de Fluorescência (FLIM) combina a resolução temporal ao conceito de microscopia, de forma que imagens de tempos de vida de fluorescência podem ser gerados. Essa técnica tem um grande potencial para aplicações clínicas uma vez que ela pode ser capaz de detectar lesões e delinear suas bordas. No entanto, FLIM requer uma instrumentação muito mais sofisticada do que a maior parte das técnicas baseadas no estado estacionário, o que cria uma dificuldade para que tais sistemas possam ser levados a ambientes clínicos. Nós reportamos a montagem, caracterização, validação e aplicação clínica de um sistema FLIM multiespectral com uma sonda manual composta de um endoscópio rígido de varredura laser. O sistema montado utiliza um laser pulsado de 355 nm como fonte de excitação e conta com três canais espectrais, visando a emissão do colágeno, do NADH e do FAD, três importantes fluoróforos endógenos. O sistema é capaz de adquirir imagens de áreas de 8,65 x 8,65 mm2 em ~ 2,4 s. Códigos em MATLAB foram escritos para processar as imagens usando um modelo biexponencial e uma abordagem modificada dos fasores. Medidas in vivo de tumores induzidos em camundongos foram realizadas para validação do sistema. O sistema também foi validado com a realização de medidas in vivo da pele de voluntários sadios. O sistema montado foi levado ao Hospital Amaral Carvalho, onde realizamos um teste clínico piloto no qual diferentes tipos de lesões de pele foram imageados in vivo em um ambiente clínico. Um contraste significante foi alcançado em tumores de queratose seborreica, doença de Bowen e carcinoma basocelular esclerodermiforme. Esses resultados indicam o potencial desta técnica para o imageamento clínico de lesões de pele.

Fluorescence based microscopy techniques have been extensively used in biological sciences. The most common approach is the steady-state fluorescence microscopy. Although the said approach is powerful, it often lacks sensitivity to detect several biochemical processes that may indicate relevant conditions of biological tissues. The fluorescence dynamics analysis not only brings intrinsic information about the tissue, but is also less sensitive to the medium scattering and absorption, and sometimes capable of distinguishing between fluorescent structures with indistinguishable spectra. The intrinsic fluorescence lifetime of biological tissues is usually affected by some clinical conditions, especially when those conditions cause or are correlated with metabolic modifications. Time-resolved spectroscopy techniques can be used to detect those modifications and may be used as a tool to improve the detection and diagnosis rate of such conditions. Fluorescence Lifetime Imaging Microscopy (FLIM) combines the temporal resolution and the microscopy concept, so fluorescence lifetime images can be generated. This technique has a great potential for clinical applications since it may be able to detected lesions and delineate its borders. However, FLIM usually demands a more sophisticated instrumentation than most techniques based on the steady-state approach, what creates a difficulty for moving such a system to a clinical setting. We report the assembly, characterization, validation, and clinical application of a multispectral FLIM system featuring a handheld probe composed of a laser scanning rigid endoscope. The assembled system uses a 355 nm short pulsed laser as excitation and has three spectral channels, targeting the emission of collagen, NADH, and FAD, which are important endogenous fluorophores. The system acquires images of 8.65 x 8.65 mm2 areas in ~ 2.4 s. MATLAB codes were written to process the images using a biexponential model and a modified phasor approach. In vivo validation measurements of tumors induced in mice were performed. The system was also validated with in vivo imaging of skin of healthy volunteers. The assembled FLIM system was moved to Hospital Amaral Carvalho, where we performed a pilot clinical study, in which different types of skin lesions were imaged in vivo in a clinical setting. A significant contrast was achieved on seborrheic keratosis, Bowen´s disease, and sclerodermiform basal cell carcinoma tumors. These results indicate the potential of this technique for clinical imaging of skin lesions. / Técnicas de microscopia baseadas em fluorescência têm sido extensamente utilizadas em ciências biológicas. A abordagem mais comum se baseia na microscopia de fluorescência de estado estacionário. Apesar de poderosa, essa abordagem frequentemente não apresenta sensibilidade suficiente para detectar diversos processos bioquímicos que podem ser indicadores de relevantes problemas em tecidos biológicos. A análise da dinâmica da fluorescência não apenas trás informações intrínsecas sobre o tecido, mas também é menos sensível a espalhamento e absorção pelo meio, além de ser capaz de distinguir entre estruturas fluorescentes com espectros indistinguíveis em alguns casos. O tempo de vida intrínseco de tecidos biológicos é normalmente afetado por condições clínicas, especialmente quando estas condições causam ou são relacionadas a modificações metabólicas. As técnicas de espectroscopia resolvidas no tempo podem detectar essas modificações e podem ser utilizadas como uma ferramenta para melhorar a detecção e o diagnóstico dessas condições. A Microscopia de Tempo de Vida de Fluorescência (FLIM) combina a resolução temporal ao conceito de microscopia, de forma que imagens de tempos de vida de fluorescência podem ser gerados. Essa técnica tem um grande potencial para aplicações clínicas uma vez que ela pode ser capaz de detectar lesões e delinear suas bordas. No entanto, FLIM requer uma instrumentação muito mais sofisticada do que a maior parte das técnicas baseadas no estado estacionário, o que cria uma dificuldade para que tais sistemas possam ser levados a ambientes clínicos. Nós reportamos a montagem, caracterização, validação e aplicação clínica de um sistema FLIM multiespectral com uma sonda manual composta de um endoscópio rígido de varredura laser. O sistema montado utiliza um laser pulsado de 355 nm como fonte de excitação e conta com três canais espectrais, visando a emissão do colágeno, do NADH e do FAD, três importantes fluoróforos endógenos. O sistema é capaz de adquirir imagens de áreas de 8,65 x 8,65 mm2 em ~ 2,4 s. Códigos em MATLAB foram escritos para processar as imagens usando um modelo biexponencial e uma abordagem modificada dos fasores. Medidas in vivo de tumores induzidos em camundongos foram realizadas para validação do sistema. O sistema também foi validado com a realização de medidas in vivo da pele de voluntários sadios. O sistema montado foi levado ao Hospital Amaral Carvalho, onde realizamos um teste clínico piloto no qual diferentes tipos de lesões de pele foram imageados in vivo em um ambiente clínico. Um contraste significante foi alcançado em tumores de queratose seborreica, doença de Bowen e carcinoma basocelular esclerodermiforme. Esses resultados indicam o potencial desta técnica para o imageamento clínico de lesões de pele.

The transmembrane protein prestin forms an integral part of the mammalian sense of hearing by providing the driving force for the electromotility of the outer hair cell, a specialized cell that resides within the cochlea. This provides the cochlea with an ability to amplify mechanical vibrations, allowing for a high degree of sensitivity and selectivity in auditory transduction. The phenomenon, driven by changes in the transmembrane potential, is thought to be the result of conformational changes in self-associating prestin oligomers. We have previously utilized Forster resonance energy transfer (FRET), by both sensitized emission and acceptor photobleach methods, to detect prestin self -association. While these methods can qualitatively confirm prestin-prestin association, determining nanoscale changes in prestin organization requires greater accuracy than either technique provides. In this thesis, a FRET methodology based on fluorescence lifetime imaging (FLIM), detected by time correlated single photon counting (TCSPC), is implemented and utilized to quantitatively measure conformational changes within prestin-prestin oligomers in response to voltage stimulus.

No description available.

Le cycle cellulaire est finement régulé dans le temps et l'espace. Nous avons abordé les aspects dynamiques des interactions que la cycline A2 entretient avec ses partenaires Cdk1, Cdk2 et l'ubiquitine au cours du cycle cellulaire, dans des lignées cellulaires humaines. A cette fin, nous avons eu recours aux approches de FRET (Förster/fluorescence resonance energy transfer) et de FLIM (fluorescence lifetime imaging microscopy). Ceci nous a permis de montrer que les formes ubiquitinylées de la cycline A2 apparaissent principalement sous forme de foyers en prométaphase et se propagent ensuite à l'ensemble de la cellule. En outre, nous avons découvert que l'autophagie participe à la dégradation de cette cycline en mitose. Nous discutons les implications de ces observations quant à un rôle éventuel de la cycline A2 au moment de la formation de l'anneau de constriction, ainsi que de la participation de l'autophagie via cette cycline, dans la réponse aux dommages à l'ADN en mitose. / The cell cycle is finely regulated in time and space. We have studied the dynamical aspect of the interactions between cyclin A2 and its partners Cdk1, Cdk2 and ubiquitin during the cell cycle, in human cell lines. To this aim, we have used FRET (Förster/fluorescence resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) techniques. We have thus shown that ubiquitylated forms of cyclin A2 are detected predominantly in foci in prometaphase, before spreading throughout the cell. Moreover, we have shown that autophagy contributes to cyclin A2 degradation in mitosis. We discuss the implications of these observations regarding a possible role of cyclin A2 when the cleavage furrow forms, and the participation of autophagy in DNA damage response in mitosis.

A terapia fotodinâmica é uma técnica que vem sendo aprimorada para o tratamento de cânceres e infecções e seu efeito terapêutico consiste na geração de espécies reativas de oxigênio que causam danos irreversíveis às células, levando-as a morte. Ela é considerada menos invasiva que outros tratamentos, podendo ser repetida diversas vezes e utilizada em conjunto com outros tratamentos sem o comprometimento do paciente. Um dos focos da terapia fotodinâmica é o tratamento de cânceres de pele melanoma devido a localização superficial das lesões e a elevada taxa de letalidade que a doença ocasiona, esta alta taxa esta relacionada a formação de tumores secundários em outras regiões do corpo que dificulta a cura. Neste trabalho buscou-se entender a interação do fotossensibilizador de segunda geração, Photodithazine, com as células de melanoma murino de linhagem B16F10. Foram analisadas a captação celular e a co-localização intracelular deste fotossensibilizador através da marcação de componentes celulares em microscopia confocal. Ensaios de viabilidade celular foram realizados para obtenção das concentrações menos citotóxicas sem e com luz, utilizando o teste de MTT. Foi realizada a caracterização do tempo de vida médio fluorescência do fotossensibilzador durante a terapia fotodinâmica em microscópio FLIM (fluorescence lifetime imaging microscopy). Os resultados indicaram que um maior tempo de incubação no escuro causa maior citotoxicidade para as concentrações acima de 0,1 mg/mL de Photodithazine. Os ensaios de terapia fotodinâmica apresentaram redução de mais de 90% de viabilidade celular para baixas concentrações de Photodithazine®, utilizando fluências acima de 2J/cm2. O tempo de vida médio de fluorescência do Photodithazine livre em solução apresentou tempo de 3,75 ns e quando ligado às células de 4,63 ns, durante a terapia fotodinâmica o tempo de vida médio de fluorescência apresentou alteração de aproximadamente 0,2 ns. Através do cálculo de coeficientes de correlação, foi observado a alta co-localização em mitocôndrias. / Photodynamic therapy is a technique manly applied in the treatment of cancers and infections. The principal compound of this therapy is the reactive oxygen species generated that react and damage various cellular components, causing cell death. Photodynamic therapy is considered minimally invasive and can be repeated several times without compromising the patient. One of the main focuses of photodynamic therapy is threatment of melanoma skin cancer due to it is superficial location of the lesions and also because the disease has a high rate of mortality, caused by the formation of secondary tumors elsewhere in the body that makes it difficult to cure. In this study, murine melanoma cells were studied in monolayer model using Photodithazine, a photosensitizer of second generation. Cellular uptake and intracellular co-localization of the photosensitizer was observed by labeling cellular components that were analyzed by confocal microscopy. Cell viability assays were performed to obtain the cytotoxic concentrations in the dark, using the MTT test. Using the less cytotoxic concentrations, photodynamic therapy trials was performed to obtain the cytotoxic fluences. Finally, the average lifetime fluorescence of photosensitizer was measured to characterize the photodynamic therapy in FLIM (fluorescence lifetime imaging microscopy). The results indicated that longer dark incubation cause greater cytotoxicity at the concentrations above 0.1 mg/mL of Photodithazine. Thereby the results of photodynamic therapy experiments observed the decreasing from over 90% cell viability at low concentrations of Photodithazine using fluences above 2 J/cm2. The average fluorescence lifetime of the free Photodithazine solution was 3.75 ns and cell-bound 4.63 ns, during the photodynamic therapy the average lifetime fluorescence changed approximately 0.2 ns. By calculating correlation coefficients, the high colocalization was observed in mitochondria.

NEDD8, molécule de la famille de l’ubiquitine est essentielle au développement, à la croissance et à la viabilité d’un organisme, de plus c’est une cible prometteuse en thérapeutique. Nous avons découvert que l’inhibiteur spécifique de la NEDDylation, MLN4924 altère la morphologie sans fragmentation et augmente la surface du nucléole de cellules humaines et de noyaux de la lignée germinale de Caenorhabditis elegans. Une approche de protéomique quantitative (SILAC) combiné à l’analyse de la production des ARNr et des ribosomes montrent que MLN4924 change la composition protéique du nucléole sans affecter l’activité transcriptionnelle de l’ARN pol I. Notre analyse montre que MLN4924 active p53 par la voie RPL11/RPL5-Mdm2 caractéristique d’un stress du nucléole. Cette étude identifie le nucléole comme une cible intéressante dans l’utilisation d’inhibiteurs de la NEDDylation et apporte un nouveau mécanisme d’activation de p53 par inhibition de la voie NEDD8.Dans une seconde étude nous avons adapté la méthode de FLIM-FRET (« Fluorescence Lifetime Imaging Microscopy – Förster Resonance Energy Transfer ») à l’étude de la compaction de la chromatine à l’échelle du nanomètre dans un organisme vivant. Le nématode Caenorhabditis elegans s’est révélé être un modèle de choix. Au sein des chromosomes méiotiques, nous avons identifié différentes régions de compaction, de niveau variable par mesure du FRET entre histones fusionnées à des protéines fluorescentes. Par une approche originale d’ARN interférence et injection d’un « extra-chromosome » nous avons défini l’architecture à une nano-échelle de différents états de l’hétérochromatine et montré que cette organisation est contrôlée par les protéines HP1 « Heterochromatin Protein 1 » et SETDB1, une protéine « H3-Lysine 9 methyl transferase ». Nous avons également montré que la compaction de l’hétérochromatine est dépendante des condensines I et II et plus particulièrement la condensine I contrôle l’état faiblement compacté de la chromatine.Nos travaux ont confirmé que C. elegans est un modèle d’intérêt majeur pour l’étude des compartiments nucléaires et parfaitement adapté pour des études pré-clinique. / The ubiquitin-like molecule NEDD8 is conserved and essential for viability, growth and development; its activation pathway is a promising target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human cells and Caenorhabditis elegans germ cells in the absence of nucleolar fragmentation. Through SILAC proteomic analysis and rRNA production, processing and ribosome profiling, we show that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of the NEDDylation pathway and provides a mechanism for p53 activation upon NEDD8 inhibition.Then we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay the nano-scale chromatin compaction in a living organism, the nematode Caenorhabditis elegans. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyl-transferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.We confirm that C. elegans is an interesting model to study nuclear signalling and perfectly adapt to be a platform for pre-clinical studies.

A terapia fotodinâmica é uma técnica que vem sendo aprimorada para o tratamento de cânceres e infecções e seu efeito terapêutico consiste na geração de espécies reativas de oxigênio que causam danos irreversíveis às células, levando-as a morte. Ela é considerada menos invasiva que outros tratamentos, podendo ser repetida diversas vezes e utilizada em conjunto com outros tratamentos sem o comprometimento do paciente. Um dos focos da terapia fotodinâmica é o tratamento de cânceres de pele melanoma devido a localização superficial das lesões e a elevada taxa de letalidade que a doença ocasiona, esta alta taxa esta relacionada a formação de tumores secundários em outras regiões do corpo que dificulta a cura. Neste trabalho buscou-se entender a interação do fotossensibilizador de segunda geração, Photodithazine, com as células de melanoma murino de linhagem B16F10. Foram analisadas a captação celular e a co-localização intracelular deste fotossensibilizador através da marcação de componentes celulares em microscopia confocal. Ensaios de viabilidade celular foram realizados para obtenção das concentrações menos citotóxicas sem e com luz, utilizando o teste de MTT. Foi realizada a caracterização do tempo de vida médio fluorescência do fotossensibilzador durante a terapia fotodinâmica em microscópio FLIM (fluorescence lifetime imaging microscopy). Os resultados indicaram que um maior tempo de incubação no escuro causa maior citotoxicidade para as concentrações acima de 0,1 mg/mL de Photodithazine. Os ensaios de terapia fotodinâmica apresentaram redução de mais de 90% de viabilidade celular para baixas concentrações de Photodithazine®, utilizando fluências acima de 2J/cm2. O tempo de vida médio de fluorescência do Photodithazine livre em solução apresentou tempo de 3,75 ns e quando ligado às células de 4,63 ns, durante a terapia fotodinâmica o tempo de vida médio de fluorescência apresentou alteração de aproximadamente 0,2 ns. Através do cálculo de coeficientes de correlação, foi observado a alta co-localização em mitocôndrias. / Photodynamic therapy is a technique manly applied in the treatment of cancers and infections. The principal compound of this therapy is the reactive oxygen species generated that react and damage various cellular components, causing cell death. Photodynamic therapy is considered minimally invasive and can be repeated several times without compromising the patient. One of the main focuses of photodynamic therapy is threatment of melanoma skin cancer due to it is superficial location of the lesions and also because the disease has a high rate of mortality, caused by the formation of secondary tumors elsewhere in the body that makes it difficult to cure. In this study, murine melanoma cells were studied in monolayer model using Photodithazine, a photosensitizer of second generation. Cellular uptake and intracellular co-localization of the photosensitizer was observed by labeling cellular components that were analyzed by confocal microscopy. Cell viability assays were performed to obtain the cytotoxic concentrations in the dark, using the MTT test. Using the less cytotoxic concentrations, photodynamic therapy trials was performed to obtain the cytotoxic fluences. Finally, the average lifetime fluorescence of photosensitizer was measured to characterize the photodynamic therapy in FLIM (fluorescence lifetime imaging microscopy). The results indicated that longer dark incubation cause greater cytotoxicity at the concentrations above 0.1 mg/mL of Photodithazine. Thereby the results of photodynamic therapy experiments observed the decreasing from over 90% cell viability at low concentrations of Photodithazine using fluences above 2 J/cm2. The average fluorescence lifetime of the free Photodithazine solution was 3.75 ns and cell-bound 4.63 ns, during the photodynamic therapy the average lifetime fluorescence changed approximately 0.2 ns. By calculating correlation coefficients, the high colocalization was observed in mitochondria.

Oxidized phospholipids (OxPLs) are known to be present in living organisms due to oxidative stress. However, the physiological function of OxPLs is still not fully understood. They have been shown to be present in many inflammatory diseases such as atherosclerosis and neurodegenerative diseases like Parkinson's and Alzheimer's disease. In this work we present the influence of two truncated OxPLs on the lateral heterogeneity of a model lipid membrane. Specifically, we studied the effect of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3- phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) on the formation of nanodomains present in giant unilamellar vesicles containing 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol and sphingomyelin. Only few techniques are capable of detecting nanometer-sized domains in the membrane with high resolution. Time resolved Förster resonance energy transfer (TR-FRET) combined with Monte Carlo (MC) simulations provide a strong tool not only to detect lateral heterogeneities but also characterize them with the resolution of 2 nm. Profound effects on the nanodomain size were observed in the presence of both studied OxPLs and differences were detected, as PGPC with a carboxylic group drives formation of larger nanodomains than POVPC...

The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.